Arresting the bad seed: HDAC3 regulates proliferation of different microglia after ischemic stroke

May combine two claims.

Deleting HDAC3 down-regulates mitosis-related genes transcriptionally, which may obstruct mitotic phase entry.

“we concluded that deleting HDAC3 down-regulated mitosis-related genes transcriptionally, which may obstruct mitotic phase entry as suggested by a previous study (34).”

• cites [34] M. J. Hoffmann, S. Meneceur, K. Hommel, W. A. Schulz, G. Niegisch, Downregulation of cell cycle and checkpoint genes by class I HDAC inhibitors limits synergism with G2/M checkpoint inhibitor MK-1775 in bladder cancer cells. Genes 12, 260 (2021). 33670166 PMC7916885 sourceDisputed

  In Results §3.4, the HDAC3-specific inhibitor RGFP966 did not significantly affect entry into M phase; the paper attributes G2 arrest instead to inhibition of HDAC1 and HDAC2. The Discussion reports some reduction in AURKB expression with RGFP966 but explicitly says it did not significantly impede progression into mitosis. The study tested inhibition, not deletion, of HDAC3. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Long-term inhibition of CSF-1R before and after stroke exacerbates the production of inflammatory mediators, leukocyte infiltration, and ischemic lesion within the early stage of injury, while short-term CSF-1R inhibition resolves inflammation and ameliorates brain damage.

“Similarly, long-term inhibition of CSF-1R before and after stroke exacerbates the production of inflammatory mediators, leukocyte infiltration, and ischemic lesion within the early stage of injury (62), while short-term CSF-1R inhibition resolves inflammation and ameliorates brain damage (63).”

• cites [62] A. Otxoa-de-Amezaga, F. Miró-Mur, J. Pedragosa, M. Gallizioli, C. Justicia, N. Gaja-Capdevila, F. Ruíz-Jaen, A. Salas-Perdomo, A. Bosch, M. Calvo, L. Márquez-Kisinousky, A. Denes, M. Gunzer, A. M. Planas, Microglial cell loss after ischemic stroke favors brain neutrophil accumulation. Acta Neuropathol. 137, 321– 341 (2019). 30580383 PMC6513908 sourceDisputed

  Split into 2 atoms — 0 confirmed, 1 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [63] E. F. Willis, K. P. A. MacDonald, Q. H. Nguyen, A. L. Garrido, E. R. Gillespie, S. B. R. Harley, P. F. Bartlett, W. A. Schroder, A. G. Yates, D. C. Anthony, S. Rose-John, M. J. Ruitenberg, J. Vukovic, Repopulating microglia promote brain repair in an IL-6-dependent manner. Cell 180, 833– 846.e16 (2020). 32142677 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

After inhibiting CSF-1R, the resident microglia display elevated proliferation and inflammation markers.

“In addition, it was reported that after inhibiting CSF-1R, the resident microglia display elevated proliferation and inflammation markers (64, 65)”

• cites [64] W.-N. Jin, S. X.-Y. Shi, Z. Li, M. Li, K. Wood, R. J. Gonzales, Q. Liu, Depletion of microglia exacerbates postischemic inflammation and brain injury. J. Cereb. Blood Flow Metab. 37, 2224– 2236 (2017). 28273719 PMC5444553 sourceDisputed

  The Results section on PLX3397 treatment reports that the CSF1R/c-Kit inhibitor reduced resident microglia by nearly 90%, rather than showing elevated microglial proliferation. The Results section on postischemic inflammation reports increased inflammatory mediators in brain tissue after microglial depletion; it does not attribute those increases to the remaining microglia. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [65] M. R. P. Elmore, A. R. Najafi, M. A. Koike, N. N. Dagher, E. E. Spangenberg, R. A. Rice, M. Kitazawa, B. Matusow, H. Nguyen, B. L. West, K. N. Green, Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380– 397 (2014). 24742461 PMC4161285 sourceDisputed

  In Results (‘CSF1R inhibitors dramatically reduce microglia numbers in the adult brain’; page unavailable), inhibition prevented an LPS-induced increase in the inflammatory marker IBA1 and markedly reduced microglial numbers. Results (‘Rapid restoration of CNS microglia following drug removal’; page unavailable) place the proliferation marker Ki67 in newly repopulating cells after inhibitor withdrawal, not in resident microglia during inhibition. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

May combine two claims.

Depleting microglia using CSF-1R inhibitors exacerbates stroke-induced neuronal excitotoxicity, leukocyte infiltration, and brain injury.

“Depleting microglia using colony-stimulating factor 1 receptor (CSF-1R) inhibitors exacerbates stroke-induced neuronal excitotoxicity, leukocyte infiltration, and brain injury (5).”

• cites [5] C. Barca, C. Foray, S. Hermann, U. Herrlinger, I. Remory, D. Laoui, M. Schäfers, O. M. Grauer, B. Zinnhardt, A. H. Jacobs, The colony stimulating factor-1 receptor (CSF-1R)-mediated regulation of microglia/macrophages as a target for neurological disorders (glioma, stroke). Front. Immunol. 12, 787307 (2021). 34950148 PMC8688767 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

HDAC3 is a stoichiometry component of both NCoR1 and SMRT.

“only HDAC3 is a stoichiometry component of both the nuclear receptor corepressor 1 (NCoR1 or NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) (8).”

• cites [8] M. J. Emmett, M. A. Lazar, Integrative regulation of physiology by histone deacetylase 3. Nat. Rev. Mol. Cell Biol. 20, 102– 115 (2019). 30390028 PMC6347506 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Pharmacological inhibition of microglial proliferation prevents progression of Alzheimer's disease-like pathology.

“prevents progression of Alzheimer’s disease–like pathology (2)”

• cites [2] A. Olmos-Alonso, S. T. T. Schetters, S. Sri, K. Askew, R. Mancuso, M. Vargas-Caballero, C. Holscher, V. H. Perry, D. Gomez-Nicola, Pharmacological targeting of CSF1R inhibits microglial proliferation and prevents the progression of Alzheimer's-like pathology. Brain 139, 891– 907 (2016). 26747862 PMC4766375 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microglia depletion using CXCR1creER:iDTR transgenic mice and diphtheria toxin inhibits neuronal degeneration and increases anti-inflammatory factors.

“Another microglia-depleting system using CXCR1creER:iDTR transgenic mice and diphtheria toxin inhibits neuronal degeneration and increases the expression level of anti-inflammatory factors instead (6).”

• cites [6] T. Li, J. Zhao, W. Xie, W. Yuan, J. Guo, S. Pang, W.-B. Gan, D. Gómez-Nicola, S. Zhang, Specific depletion of resident microglia in the early stage of stroke reduces cerebral ischemic damage. J. Neuroinflammation 18, 81 (2021). 33757565 PMC7986495 sourcePartly supported

  The Results report fewer degenerating neurons and increased mRNA expression of anti-inflammatory factors after microglial depletion in mice three days after ischemic stroke. However, the Methods identify the mice as CX3CR1CreER:R26iDTR, not CXCR1creER:iDTR, and specify that depletion required tamoxifen followed by diphtheria toxin. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Pharmacological inhibition of microglial proliferation attenuates neuropathic pain hypersensitivities.

“Pharmacological inhibition of microglial proliferation attenuates neuropathic pain hypersensitivities (1)”

• cites [1] N. Gu, J. Peng, M. Murugan, X. Wang, U. B. Eyo, D. Sun, Y. Ren, E. DiCicco-Bloom, W. Young, H. Dong, L.-J. Wu, Spinal microgliosis due to resident microglial proliferation is required for pain hypersensitivity after peripheral nerve injury. Cell Rep. 16, 605– 614 (2016). 27373153 PMC4956495 sourceSupported

May combine two claims.

Significant infiltration of myeloid cells into the brain occurs, and these cells likely express similar levels of Iba1, CD16, and Arg1 as microglia.

“because of the significant infiltration of myeloid cells into the brain, which likely express similar levels of Iba1, CD16, and Arg1 as microglia (35–37)”

• cites [35] G. E. Holder, C. M. McGary, E. M. Johnson, R. Zheng, V. T. John, C. Sugimoto, M. J. Kuroda, W. K. Kim, Expression of the mannose receptor CD206 in HIV and SIV encephalitis: A phenotypic switch of brain perivascular macrophages with virus infection. J. Neuroimmune Pharmacol. 9, 716– 726 (2014). 25146376 PMC4364002 sourcePartly supported

  The Introduction describes macrophage accumulation in HIV encephalitis, and the Results report CD16 expression on perivascular macrophages and increasing numbers of Iba1+ cells in developing lesions. The Discussion also notes Iba1 on lesional macrophages. The paper does not report Arg1 expression or show that infiltrating cells express any of these markers at levels similar to microglia; its findings are specific to HIV/SIV encephalitis. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [36] G. Faraco, L. Park, J. Anrather, C. Iadecola, Brain perivascular macrophages: Characterization and functional roles in health and disease. J. Mol. Med. 95, 1143– 1152 (2017). 28782084 PMC5812456 sourcePartly supported

  The characterization section reports that brain-resident myeloid cells express Iba-1, but findings on whether perivascular macrophages express levels similar to microglia are conflicting. The section on macrophage origins describes monocyte entry into brain tissue after irradiation, while reporting that it did not occur when the brain was shielded; it does not establish significant infiltration generally. The passages do not support comparable CD16 or Arg1 expression. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [37] K. Bartalska, V. Hübschmann, M. Korkut-Demirbaş, R. J. A. Cubero, A. Venturino, K. Rössler, T. Czech, S. Siegert, A systematic characterization of microglia-like cell occurrence during retinal organoid differentiation. iScience 25, 104580 (2022). 35789843 PMC9250027 sourcePartly supported

  The Introduction and Discussion describe microglia entering the human embryonic cortex, and the Results identify IBA1-positive microglia-like cells. The paper does not establish significant infiltration of myeloid cells into the brain or compare their Iba1, CD16, and Arg1 expression levels with microglia; its organoid cells did not extensively populate cerebral compartments. Page numbers were not available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

Microglial expansion primarily occurs on day 3 after tFCI, while it remains relatively quiescent on day 1 after tFCI.

“This result was aligned with previous studies, which indicates that microglial expansion primarily occurs on day 3 after tFCI, while it remains relatively quiescent on day 1 after tFCI (21).”

• cites [21] T. Li, S. Pang, Y. Yu, X. Wu, J. Guo, S. Zhang, Proliferation of parenchymal microglia is the main source of microgliosis after ischaemic stroke. Brain 136, 3578– 3588 (2013). 24154617 sourcePartly supported

  The Figure 5 caption reports proliferating microglia at day 3, supporting expansion by that time. But the ‘Activation and expansion’ section reports a continuous increase in reactive microglial density from day 1 and a peak in reactive-microglial volume on day 4; it does not establish that day 1 was quiescent or that expansion occurred primarily on day 3. The Methods describe a photothrombosis stroke model, not tFCI. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Blocking microglial proliferation through diphtheria toxin administration in CX3CR1creER:R26iDTR mice significantly decreases inflammatory cells and alters immune profiles in the context of stroke.

“previous studies have revealed that blocking microglial proliferation through diphtheria toxin administration in CX3CR1creER:R26iDTR mice significantly decreased inflammatory cells and altered the immune profiles in the context of stroke, suggesting a potential connection between proliferation and inflammatory response in microglia (6).”

• cites [6] T. Li, J. Zhao, W. Xie, W. Yuan, J. Guo, S. Pang, W.-B. Gan, D. Gómez-Nicola, S. Zhang, Specific depletion of resident microglia in the early stage of stroke reduces cerebral ischemic damage. J. Neuroinflammation 18, 81 (2021). 33757565 PMC7986495 sourcePartly supported

  The Results sections “Microglial depletion decreased inflammatory cells” and “Depletion of microglia altered the immune microenvironment in ischemic tissue” report fewer iNOS+ cells and changes in inflammatory-factor expression after stroke. But the Methods and Results describe tamoxifen-plus-diphtheria-toxin treatment as depleting resident microglia, not specifically blocking their proliferation. No page numbers are provided in our copy. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Knockdown or overexpression of PU.1 in the BV2 rodent microglia cell line identifies a hub of neuroinflammation-related differentially expressed genes.

“knockdown or overexpression of PU.1 in the BV2 rodent microglia cell line identified a hub of neuroinflammation-related differentially expressed genes (DEGs) (15).”

• cites [15] A. A. Pimenova, M. Herbinet, I. Gupta, S. I. Machlovi, K. R. Bowles, E. Marcora, A. M. Goate, Alzheimer's-associated PU.1 expression levels regulate microglial inflammatory response. Neurobiol. Dis. 148, 105217 (2021). 33301878 PMC7808757 sourcePartly supported

  The Results section reports RNA sequencing after PU.1 knockdown and overexpression in BV2 mouse microglial cells, identifying differentially expressed genes, including genes involved in immune responses. It does not identify a hub of neuroinflammation-related DEGs; that network-level characterization goes beyond the reported findings (page not available in our copy). (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 is a master regulator of multiple cell cycle genes in hematopoietic stem cells.

“It was also verified to be a master regulator of multiple cell cycle genes in hematopoietic stem cells from which macrophages develop (14).”

• cites [14] P. B. Staber, P. Zhang, M. Ye, R. S. Welner, C. Nombela-Arrieta, C. Bach, M. Kerenyi, B. A. Bartholdy, H. Zhang, M. Alberich-Jordà, S. Lee, H. Yang, F. Ng, J. Zhang, M. Leddin, L. E. Silberstein, G. Hoefler, S. H. Orkin, B. Göttgens, F. Rosenbauer, G. Huang, D. G. Tenen, Sustained PU.1 levels balance cell-cycle regulators to prevent exhaustion of adult hematopoietic stem cells. Mol. Cell 49, 934– 946 (2013). 23395001 PMC3644723 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Attenuating PU.1 can potentially restrain microglia-mediated neuroinflammation in various neurological disorders.

“In various neurological disorders, attenuating PU.1 can potentially restrain microglia-mediated neuroinflammation (16).”

• cites [16] E. D. Ponomarev, T. Veremeyko, N. Barteneva, A. M. Krichevsky, H. L. Weiner, MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α-PU.1 pathway. Nat. Med. 17, 64– 70 (2011). 21131957 PMC3044940 sourcePartly supported

  The Results (‘miR-124 downregulates C/EBP-α and PU.1’; page unavailable) show that miR-124 indirectly lowers PU.1 and macrophage activation markers. The Results (‘miR-124 inhibits EAE and reduces CNS inflammation’; page unavailable) report reduced activated microglia and CNS inflammation in treated EAE mice. The paper does not establish that attenuating PU.1 itself restrains microglia-mediated neuroinflammation across various neurological disorders; its intervention was miR-124, tested for inflammation in EAE. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

Peripheral myeloid cells are continuously replaced by CX3CR1− progenitors in the bone marrow, whereas microglia self-renew and retain CX3CR1 expression during the lifetime.

“peripheral myeloid cells were continuously replaced by CX3CR1− progenitors in the bone marrow so that the original HDAC3-knockout macrophage population was almost completely renewed within 30 days, whereas microglia underwent self-renewal and retained CX3CR1 expression during the lifetime (20, 21).”

• cites [20] M. S. Mendes, L. Le, J. Atlas, Z. Brehm, A. Ladron-de-Guevara, E. Matei, C. Lamantia, M. N. McCall, A. K. Majewska, The role of P2Y12 in the kinetics of microglial self-renewal and maturation in the adult visual cortex in vivo. eLife 10, e61173 (2021). 34250902 PMC8341987 sourcePartly supported

  The Introduction says microglia are maintained throughout adult life and self-renew slowly; the Results also show residual microglia dividing to replenish the visual cortex. The paper does not establish that peripheral myeloid cells are continuously replaced by CX3CR1− bone-marrow progenitors, or that microglia retain CX3CR1 expression throughout life. No page numbers are available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [21] T. Li, S. Pang, Y. Yu, X. Wu, J. Guo, S. Zhang, Proliferation of parenchymal microglia is the main source of microgliosis after ischaemic stroke. Brain 136, 3578– 3588 (2013). 24154617 sourcePartly supported

  The section on proliferation of local resident cells reports CX3CR1-reporter-positive microglia dividing after stroke, supporting microglial self-renewal in that setting. It does not establish that microglia retain CX3CR1 throughout life or that peripheral myeloid cells are continuously replaced by CX3CR1-negative bone-marrow progenitors. It also does not address renewal of an HDAC3-knockout macrophage population within 30 days. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

In microglia, the dynamic modulation of the epigenetic landscape guided the reprogramming of the transcriptional profiles.

“In microglia, the dynamic modulation of the epigenetic landscape guided the reprogramming of the transcriptional profiles (38).”

• cites [38] M. R. Corces, A. Shcherbina, S. Kundu, M. J. Gloudemans, L. Frésard, J. M. Granja, B. H. Louie, T. Eulalio, S. Shams, S. T. Bagdatli, M. R. Mumbach, B. Liu, K. S. Montine, W. J. Greenleaf, A. Kundaje, S. B. Montgomery, H. Y. Chang, T. J. Montine, Single-cell epigenomic analyses implicate candidate causal variants at inherited risk loci for Alzheimer's and Parkinson's diseases. Nat. Genet. 52, 1158– 1168 (2020). 33106633 PMC7606627 sourceCould not verify

  The Results section on single-cell ATAC-seq identifies microglia-specific chromatin accessibility and enrichment of SPI1 binding, but does not describe dynamic epigenetic modulation or reprogramming of microglial transcriptional profiles. The Results section on MAPT links epigenetic differences to gene expression in a different context. Searches for microglial epigenetic changes and transcriptional reprogramming found no passage addressing the claimed relationship; the indexed passages provide no page numbers. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

HDAC3 acts at a subset of PU.1-defined macrophage-specific enhancers and thereby restricts histone marks at many IL-4-targeted genes.

“First, the reason may be that HDAC3 acts at a subset of PU.1-defined macrophage-specific enhancers and thereby restricts histone marks at many interleukin-4 (IL-4)–targeted genes (41).”

• cites [41] S. E. Mullican, C. A. Gaddis, T. Alenghat, M. G. Nair, P. R. Giacomin, L. J. Everett, D. Feng, D. J. Steger, J. Schug, D. Artis, M. A. Lazar, Histone deacetylase 3 is an epigenomic brake in macrophage alternative activation. Genes Dev. 25, 2480– 2488 (2011). 22156208 PMC3243058 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Mullican et al. revealed that HDAC3 in BMDMs localizes to a subset of PU.1-defined macrophage enhancers, suggesting that PU.1 likely facilitates adjacent binding of nuclear receptors.

“Similar to our findings, Mullican et al. (41) revealed that HDAC3 in bone marrow–derived macrophages (BMDMs) localizes to a subset of PU.1-defined macrophage enhancers, suggesting that PU.1 likely facilitates adjacent binding of nuclear receptors.”

• cites [41] S. E. Mullican, C. A. Gaddis, T. Alenghat, M. G. Nair, P. R. Giacomin, L. J. Everett, D. Feng, D. J. Steger, J. Schug, D. Artis, M. A. Lazar, Histone deacetylase 3 is an epigenomic brake in macrophage alternative activation. Genes Dev. 25, 2480– 2488 (2011). 22156208 PMC3243058 sourcePartly supported

  In “HDAC3 localizes to a subset of Pu.1-defined macrophage-specific enhancers” (Fig. 3; page not provided), the paper reports that HDAC3 binds mainly at a subset of PU.1 sites in BMDMs. It says PU.1 prepares enhancers for signal-specific transcription factors, but does not establish that PU.1 facilitates adjacent nuclear-receptor binding; nuclear-receptor motifs were not among the top 10 enriched motifs at these HDAC3 sites. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 is well known for its pivotal role in microglial development and inflammation.

“which is well known for its pivotal role in microglial development and inflammation (Fig. 7, D to F) (15, 40).”

• cites [15] A. A. Pimenova, M. Herbinet, I. Gupta, S. I. Machlovi, K. R. Bowles, E. Marcora, A. M. Goate, Alzheimer's-associated PU.1 expression levels regulate microglial inflammatory response. Neurobiol. Dis. 148, 105217 (2021). 33301878 PMC7808757 sourcePartly supported

  The Discussion (page unavailable) reports that changing PU.1 expression alters microglial inflammatory responses, supporting the inflammation part of the claim. It also says microglial proliferation depends on signaling through PU.1, but does not directly establish a pivotal role in microglial development. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [40] A. M. Smith, H. M. Gibbons, R. L. Oldfield, P. M. Bergin, E. W. Mee, R. L. M. Faull, M. Dragunow, The transcription factor PU.1 is critical for viability and function of human brain microglia. Glia 61, 929– 942 (2013). 23483680 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In the context of Alzheimer's disease, PU.1 is vital for Aβ clearance and the inflammatory response of microglia.

“In the context of Alzheimer’s disease, PU.1 is vital for Aβ clearance (56) and the inflammatory response of microglia (15).”

• cites [56] S.-F. Lau, C. Chen, W.-Y. Fu, J. Y. Qu, T. H. Cheung, A. K. Y. Fu, N. Y. Ip, IL-33-PU.1 transcriptome reprogramming drives functional state transition and clearance activity of microglia in Alzheimer's disease. Cell Rep. 31, 107530 (2020). 32320664 sourceNot checked — no full text of the source
• cites [15] A. A. Pimenova, M. Herbinet, I. Gupta, S. I. Machlovi, K. R. Bowles, E. Marcora, A. M. Goate, Alzheimer's-associated PU.1 expression levels regulate microglial inflammatory response. Neurobiol. Dis. 148, 105217 (2021). 33301878 PMC7808757 sourceSupported

May combine two claims.

Many DARs were not located near the DEGs, which could be attributed to epigenetic regulation via long-range interaction so that a DAR can regulate distant genes rather than the closest gene only.

“Of note, in our study, many DARs were not located near the DEGs, making it hard to explain why those up-regulated genes had their accessible regions closed, which could be attributed to the epigenetic regulation via long-range interaction so that a DAR can regulate distant genes rather than the closest gene only (42).”

• cites [42] A. Sanyal, B. R. Lajoie, G. Jain, J. Dekker, The long-range interaction landscape of gene promoters. Nature 489, 109– 113 (2012). 22955621 PMC3555147 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A recent study found novel AAV variants capable of delivering genetic payloads into microglia in vitro and in vivo without activating microglia with higher efficiency than generating transgene mouse model or conventional rAAVs.

“A recent study found novel AAV variants that are capable of delivering various genetic payloads into microglia in vitro and in vivo without activating microglia with higher efficiency than generating transgene mouse model or conventional rAAVs (20).”

• cites [20] M. S. Mendes, L. Le, J. Atlas, Z. Brehm, A. Ladron-de-Guevara, E. Matei, C. Lamantia, M. N. McCall, A. K. Majewska, The role of P2Y12 in the kinetics of microglial self-renewal and maturation in the adult visual cortex in vivo. eLife 10, e61173 (2021). 34250902 PMC8341987 sourceCould not verify

  The Materials and methods (Experimental animals; page unavailable) describe CX3CR1-GFP reporter and P2Y12-KO mice, while the Results (In vivo imaging; page unavailable) describe tracking microglia in those mice. Searches for AAV variants, viral-vector delivery, and microglial transduction found no passage addressing the claimed payload delivery or efficiency comparison. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

High-throughput screening of 1280 FDA-approved compounds identified vorinostat as a candidate drug for attenuating PU.1 level, which mimics partial effects of PU.1 knockdown.

“High-throughput screening of 1280 US Food and Drug Administration–approved compounds also identified an HDAC inhibitor, vorinostat, as a candidate drug for attenuating PU.1 level, which is then found to mimic partial effects of PU.1 knockdown (43).”

• cites [43] J. Rustenhoven, A. M. Smith, L. C. Smyth, D. Jansson, E. L. Scotter, M. E. V. Swanson, M. Aalderink, N. Coppieters, P. Narayan, R. Handley, C. Overall, T. I. H. Park, P. Schweder, P. Heppner, M. A. Curtis, R. L. M. Faull, M. Dragunow, PU.1 regulates Alzheimer's disease-associated genes in primary human microglia. Mol. Neurodegener. 13, 44 (2018). 30124174 PMC6102813 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

In Hela cells, knockdown of HDAC3 induces a G1-S transition defect.

“while in Hela cells, knockdown of HDAC3 induces a G1-S transition defect (31).”

• cites [31] M. Vidal-Laliena, E. Gallastegui, F. Mateo, M. Martínez-Balbás, M. J. Pujol, O. Bachs, Histone deacetylase 3 regulates cyclin A stability. J. Biol. Chem. 288, 21096– 21104 (2013). 23760262 PMC3774376 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

In the context of ischemic stroke, it is the noncanonical deacetylase-independent way by which deleting HDAC3 represses gene expression.

“Thus, it could be presumed that in the context of ischemic stroke, it is the noncanonical deacetylase-independent way (27) by which deleting HDAC3 represses gene expression.”

• cites [27] H. C. B. Nguyen, M. Adlanmerini, A. K. Hauck, M. A. Lazar, Dichotomous engagement of HDAC3 activity governs inflammatory responses. Nature 584, 286– 290 (2020). 32760002 PMC7725280 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

The latter role is also found in the model of experimental autoimmune encephalomyelitis (EAE) and optic nerve injury.

“The latter role is also found in the model of experimental autoimmune encephalomyelitis (EAE) (16) and optic nerve injury (57).”

• cites [16] E. D. Ponomarev, T. Veremeyko, N. Barteneva, A. M. Krichevsky, H. L. Weiner, MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α-PU.1 pathway. Nat. Med. 17, 64– 70 (2011). 21131957 PMC3044940 sourceSupported
• cites [57] N. Zhou, K. Liu, Y. Sun, Y. Cao, J. Yang, Transcriptional mechanism of IRF8 and PU.1 governs microglial activation in neurodegenerative condition. Cell 10, 87– 103 (2019). PMC6340890 30484118 sourceSupported

May combine two claims.

HDAC3 inhibition intervened in the progressions of microglia cell cycles probably in an elusive way more or less independent of posttranslational modulation of cyclins or CDKs as shown by RNA-seq results and distinct from other cell types.

“Our study revealed that HDAC3 inhibition intervened in the progressions of microglia cell cycles probably in an elusive way more or less independent of posttranslational modulation of cyclins or CDKs as shown by RNA-seq results and distinct from other cell types (30).”

• cites [30] Y. Jiang, J. Hsieh, HDAC3 controls gap 2/mitosis progression in adult neural stem/progenitor cells by regulating CDK1 levels. Proc. Natl. Acad. Sci. U.S.A. 111, 13541– 13546 (2014). 25161285 PMC4169927 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Loss of HDAC3 in hematopoietic progenitor cells blocks only the S phase progression.

“Loss of HDAC3 in hematopoietic progenitor cells blocks only the S phase progression (45)”

• cites [45] A. R. Summers, M. A. Fischer, K. R. Stengel, Y. Zhao, J. F. Kaiser, C. E. Wells, A. Hunt, S. Bhaskara, J. W. Luzwick, S. Sampathi, X. Chen, M. A. Thompson, D. Cortez, S. W. Hiebert, HDAC3 is essential for DNA replication in hematopoietic progenitor cells. J. Clin. Invest. 123, 3112– 3123 (2013). 23921131 PMC3696547 sourcePartly supported

  The section “Inactivation of Hdac3 impairs S phase progression of MPP cells” reports increased S-phase labeling and shorter DNA-replication tracks in Hdac3-null stem/progenitor cells. It supports impaired S-phase progression, but does not establish a complete block or that S phase is the only phase affected. No page number is available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

In a preventive study, after depleting microglia by blocking CSF-1R for 21 days before tFCI, above 90% of microglia are eliminated, leading to increased brain injury, enhanced excitotoxicity, and altered neuronal activity.

“In a preventive study, after depleting microglia by blocking CSF-1R for 21 days before tFCI, above 90% of microglia are eliminated, leading to increased brain injury, enhanced excitotoxicity, and altered neuronal activity (61).”

• cites [61] G. Szalay, B. Martinecz, N. Lénárt, Z. Környei, B. Orsolits, L. Judák, E. Császár, R. Fekete, B. L. West, G. Katona, B. Rózsa, Á. Dénes, Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. Nat. Commun. 7, 11499 (2016). 27139776 PMC4857403 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 is known to be a vital transcription factor to regulate macrophage proliferation and differentiation as well as microgliogenesis in the developing brain.

“PU.1 is known to be a vital transcription factor to regulate macrophage proliferation (54) and differentiation (55) as well as microgliogenesis (13) in the developing brain.”

• cites [54] A. Celada, F. E. Borràs, C. Soler, J. Lloberas, M. Klemsz, C. van Beveren, S. McKercher, R. A. Maki, The transcription factor PU.1 is involved in macrophage proliferation. J. Exp. Med. 184, 61– 69 (1996). 8691150 PMC2192661 sourceSupported
• cites [55] G. Li, W. Hao, W. Hu, Transcription factor PU.1 and immune cell differentiation (Review). Int. J. Mol. Med. 46, 1943– 1950 (2020). 33125129 sourceCould not verify

  The citation to this source is attached to microgliogenesis in the developing brain. Section 4, “Role of PU.1 in macrophage differentiation” (page unavailable), discusses macrophage differentiation, but the passages retrieved for “microgliogenesis in the developing brain” and “microglia brain development PU.1” do not address microgliogenesis or brain development. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [13] K. Kierdorf, D. Erny, T. Goldmann, V. Sander, C. Schulz, E. G. Perdiguero, P. Wieghofer, A. Heinrich, P. Riemke, C. Hölscher, D. N. Müller, B. Luckow, T. Brocker, K. Debowski, G. Fritz, G. Opdenakker, A. Diefenbach, K. Biber, M. Heikenwalder, F. Geissmann, F. Rosenbauer, M. Prinz, Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat. Neurosci. 16, 273– 280 (2013). 23334579 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Loss of HDAC3 impairs the activation of LPS-stimulated genes, which is attributable to the reduction of IFN-β expression.

“In previous studies, loss of HDAC3 impairs the activation of LPS-stimulated genes, which is attributable to the reduction of interferon-β (IFN-β) expression (58).”

• cites [58] X. Chen, I. Barozzi, A. Termanini, E. Prosperini, A. Recchiuti, J. Dalli, F. Mietton, G. Matteoli, S. Hiebert, G. Natoli, Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages. Proc. Natl. Acad. Sci. U.S.A. 109, E2865– E2874 (2012). 22802645 PMC3479529 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

In LPS-treated BMDMs, the most enriched motif at HDAC3-bound regions also matches the binding specificity of PU.1.

“In lipopolysaccharide (LPS)–treated BMDMs, the most enriched motif at HDAC3-bound regions also matches the binding specificity of PU.1 (27).”

• cites [27] H. C. B. Nguyen, M. Adlanmerini, A. K. Hauck, M. A. Lazar, Dichotomous engagement of HDAC3 activity governs inflammatory responses. Nature 584, 286– 290 (2020). 32760002 PMC7725280 sourcePartly supported

  The main text (page unavailable) says that motif analysis of HDAC3-bound, LPS-stimulated enhancers revealed a motif bound by PU.1. Extended Data Figure 2h describes the top enriched motifs at HDAC3-bound regions, but the available text does not establish that the PU.1 motif was the single most enriched. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

In the nervous system, HDAC3 controls G2-M phase progression in adult neural stem/progenitor cells by posttranslational stabilization of the G2-M-dependent CDK1.

“In the nervous system, HDAC3 controls G2-M phase progression in adult neural stem/progenitor cells by posttranslational stabilization of the G2-M–dependent CDK1 (30).”

• cites [30] Y. Jiang, J. Hsieh, HDAC3 controls gap 2/mitosis progression in adult neural stem/progenitor cells by regulating CDK1 levels. Proc. Natl. Acad. Sci. U.S.A. 111, 13541– 13546 (2014). 25161285 PMC4169927 sourceSupported

May combine two claims.

Treating LPS-induced macrophages with PEG-A1 negatively regulated the expression of HDAC3, highlighting a potential bidirectional regulatory interaction between Arg1 and HDAC3.

“Treating LPS-induced macrophages with pegylated arginase 1 (PEG-A1) negatively regulated the expression of HDAC3, highlighting a potential bidirectional regulatory interaction between Arg1 and HDAC3.”

• cites [19] E. Shosha, R. A. Shahror, C. A. Morris, Z. Xu, R. Lucas, M. E. McGee-Lawrence, N. J. Rusch, R. B. Caldwell, A. Y. Fouda, The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: Implications for retinal ischemic injury. Cell Death Dis. 14, 621 (2023). 37735154 PMC10514323 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

A previous study reported that HDAC3-deficent microglia shift from a proinflammatory phenotype to an inflammation-resolving phenotype with a reduction of microglia number in a mouse model of TBI.

“A previous study reported that HDAC3-deficent microglia shift from a proinflammatory phenotype to an inflammation-resolving phenotype with a reduction of microglia number in a mouse model of TBI (10).”

• cites [10] Y. Zhao, H. Mu, Y. Huang, S. Li, Y. Wang, R. A. Stetler, M. V. L. Bennett, C. E. Dixon, J. Chen, Y. Shi, Microglia-specific deletion of histone deacetylase 3 promotes inflammation resolution, white matter integrity, and functional recovery in a mouse model of traumatic brain injury. J. Neuroinflammation 19, 201 (2022). 35933343 PMC9357327 sourcePartly supported

  The Results section (Fig. 2E–F) reports a shift from proinflammatory to inflammation-resolving microglia after TBI. It also says HDAC3 deletion did not change total microglial numbers in the examined regions. The Discussion (Fig. 1F–G and Fig. 2E) places the reduction in microglial numbers in the uninjured brain and says numbers rebounded after TBI. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

In a model of demyelination, proliferative microglia cluster as a population (Birc5+) independent of other pathological microglia in scRNA-seq.

“While a previous study reported that in a model of demyelination, proliferative microglia cluster as a population (Birc5+) independent of other pathological microglia in single-cell RNA sequencing (scRNA-seq) (67)”

• cites [67] T. R. Hammond, C. Dufort, L. Dissing-Olesen, S. Giera, A. Young, A. Wysoker, A. J. Walker, F. Gergits, M. Segel, J. Nemesh, S. E. Marsh, A. Saunders, E. Macosko, F. Ginhoux, J. Chen, R. J. M. Franklin, X. Piao, S. A. McCarroll, B. Stevens, Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes. Immunity 50, 253– 271.e6 (2019). 30471926 PMC6655561 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Recent investigations have identified Arg1+ microglia as key regulators of postnatal development and reducers of Aβ plaque deposition.

“Recent investigations have identified Arg1+ microglia as key regulators of postnatal development (70) and reducers of Aβ plaque deposition (71).”

• cites [70] V. Stratoulias, R. Ruiz, S. Kanatani, A. M. Osman, L. Keane, J. A. Armengol, A. Rodríguez-Moreno, A. N. Murgoci, I. García-Domínguez, I. Alonso-Bellido, F. González Ibáñez, K. Picard, G. Vázquez-Cabrera, M. Posada-Pérez, N. Vernoux, D. Tejera, K. Grabert, M. Cheray, P. González-Rodríguez, E. M. Pérez-Villegas, I. Martínez-Gallego, A. Lastra-Romero, D. Brodin, J. Avila-Cariño, Y. Cao, M. Airavaara, P. Uhlén, M. T. Heneka, M. Tremblay, K. Blomgren, J. L. Venero, B. Joseph, ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse b sourceSupported
• cites [71] J. D. Cherry, J. A. Olschowka, M. K. O'Banion, Arginase 1+ microglia reduce Aβ plaque deposition during IL-1β-dependent neuroinflammation. J. Neuroinflammation 12, 203 (2015). 26538310 PMC4634600 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Treatment with Arg1 collaborated with ornithine decarboxylase enzyme was found to protect against retinal ischemic injury.

“Furthermore, treatment with Arg1 collaborated with ornithine decarboxylase enzyme was found to protect against retinal ischemic injury (19).”

• cites [19] E. Shosha, R. A. Shahror, C. A. Morris, Z. Xu, R. Lucas, M. E. McGee-Lawrence, N. J. Rusch, R. B. Caldwell, A. Y. Fouda, The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: Implications for retinal ischemic injury. Cell Death Dis. 14, 621 (2023). 37735154 PMC10514323 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Stat6/Arg1 axis also orchestrated microglia/macrophage efferocytosis and inflammation resolution after stroke.

“Stat6/Arg1 axis also orchestrated microglia/macrophage efferocytosis and inflammation resolution after stroke (72).”

• cites [72] W. Cai, X. Dai, J. Chen, J. Zhao, M. Xu, L. Zhang, B. Yang, W. Zhang, M. Rocha, T. Nakao, J. Kofler, Y. Shi, R. A. Stetler, X. Hu, J. Chen, STAT6/Arg1 promotes microglia/macrophage efferocytosis and inflammation resolution in stroke mice. JCI Insight 4, e131355 (2019). 31619589 PMC6824303 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Using scRNA-seq, a proliferative subtype controlled by HDAC3 is also unveiled in the context of spinal cord injury.

“Recently, using scRNA-seq, a proliferative subtype controlled by HDAC3 is also unveiled in the context of spinal cord injury (7).”

• cites [7] S. Wahane, X. Zhou, X. Zhou, L. Guo, M.-S. Friedl, M. Kluge, A. Ramakrishnan, L. Shen, C. C. Friedel, B. Zhang, R. H. Friedel, H. Zou, Diversified transcriptional responses of myeloid and glial cells in spinal cord injury shaped by HDAC3 activity. Sci. Adv. 7, eabd8811 (2021). 33637528 PMC7909890 sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

The presence of HDAC3-deficient BAMs raised the possibility of their influence on microglial characteristics through direct contact or paracrine signaling pathways.

“While our focus was primarily on microglia, the presence of HDAC3-deficient BAMs raised the possibility of their influence on microglial characteristics through direct contact or paracrine signaling pathways (74) given a degree of heterogeneity of BAMs as reported in recent studies (69).”

• cites [74] S. De Schepper, J. Z. Ge, G. Crowley, L. S. S. Ferreira, D. Garceau, C. E. Toomey, D. Sokolova, J. Rueda-Carrasco, S.-H. Shin, J.-S. Kim, T. Childs, T. Lashley, J. J. Burden, M. Sasner, C. Sala Frigerio, S. Jung, S. Hong, Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer's disease. Nat. Neurosci. 26, 406– 415 (2023). 36747024 PMC9991912 sourcePartly supported

  The Main and Discussion describe potential crosstalk between perivascular macrophages and microglia, including paracrine signaling that could affect microglial phagocytosis. They do not examine HDAC3-deficient BAMs or identify direct contact as a mechanism; the HDAC3-deficient cells are the citing authors’ own context. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [69] M. J. C. Jordão, R. Sankowski, S. M. Brendecke, G. Sagar, Y. H. Locatelli, T. L. Tai, E. Tay, S. Schramm, N. Armbruster, O. Hagemeyer, D. Groß, Ö. Mai, T. Çiçek, M. Falk, D. Kerschensteiner, M. P. Grün, Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363, eaat7554 (2019). 30679343 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Direct inhibition of microglial mitosis with AraC fosters microglial development of a proinflammatory phenotype during chronic degeneration.

“Similarly, direct inhibition of microglial mitosis with AraC fosters microglial development of a proinflammatory phenotype during chronic degeneration (66).”

• cites [66] D. Gómez-Nicola, N. L. Fransen, S. Suzzi, V. H. Perry, Regulation of microglial proliferation during chronic neurodegeneration. J. Neurosci. 33, 2481– 2493 (2013). 23392676 PMC6619184 sourceSupported

May combine two claims.

HDAC3-miKO improves myelin loss and axon degeneration in white matter at 35 days after tFCI.

“HDAC3-miKO remarkably improved myelin loss 35 days after stroke in the striatum and the EC, as well as ameliorated axon degeneration 35 days after stroke in the striatum, EC, and cortex (Fig. 3, B and D)”

May combine two claims.

White matter integrity (SMI32/MBP) and axon degeneration correlate with sensorimotor behavior outcome after tFCI.

“Either the MBP intensity or the axon degeneration in the two tFCI groups was highly correlated with the sensorimotor behavior outcome assessed on the last day before euthanasia, including performance in the adhesive removal test and the rotarod test (Fig. 3G, left).”

May combine two claims.

HDAC3-miKO significantly decreases expression of most genes in G2-M phase or M phase but does not change expression of genes in G1-S phase in microglia after tFCI.

“HDAC3-miKO significantly decreased the expression of most of the genes present in G2-M phase or M phase but did not change the level of genes present in G1-S phase, more directly confirming blockade of G2-M transition induced by HDAC3 loss.”

May combine two claims.

HDAC3-deficient microglia after tFCI exhibit decreased proinflammatory gene expression and increased anti-inflammatory gene expression.

“HDAC3-deficient microglia exhibited a distinct inflammatory expression profile following stroke with decreased proinflammatory gene expression and increased anti-inflammatory gene expression, while these genes were all elevated after tFCI (WT-tFCI versus WT-Sham) (Fig. 4D).”

May combine two claims.

HDAC3-miKO reduces the number of proliferating (Ki67+) microglia but does not affect the number of G0-phase microglia after tFCI.

“HDAC3 absence reduced the number of proliferating microglia but did not affect the number of G0-phase microglia after tFCI (Fig. 5F) so that more HDAC3-deficent microglia rested in the G0 phase compared to WT microglia (Fig. 5G).”

May combine two claims.

HDAC3-miKO specifically inhibits proliferation of proinflammatory microglia without affecting anti-inflammatory microglia.

“HDAC3-miKO specifically inhibited proliferation of proinflammatory microglia without affecting anti-inflammatory microglia, preventing microglial transition to a proinflammatory state.”

May combine two claims.

HDAC3-deficient microglia show increased percentage of cells in S phase and G2-M phase and decreased percentage in G0-G1 phase after tFCI.

“HDAC3-deficient microglia showed increased percentage of cells in S phase and G2-M phase but decreased percentage of cells in G0-G1 phase (Fig. 5I)”

May combine two claims.

In Sham-operated groups, CD16/Arg1 expression was too low to detect, and no EdU+ microglia were detected.

“In Sham-operated groups, CD16/Arg1 expression was too low to detect, and no EdU+ microglia were detected (fig. S8, A and B).”

May combine two claims.

Hdac3 mRNA is almost eliminated in microglia but not affected in other brain cells in HDAC3-miKO mice.

“quantification of Hdac3 mRNA expression in these FACS-isolated microglia revealed that Hdac3 was almost eliminated in microglia, while Hdac3 expression in other brain cells was not affected (Fig. 1G, blue background).”

May combine two claims.

For miKO-tFCI versus WT-tFCI, 38.71% of closed DARs were located in the promoter region, and 31.37% were located in the intron region.

“For miKO-tFCI versus WT-tFCI, only the distribution of closed DARs was measured because there were too few open DARs; 38.71% of closed DARs were located in the promoter region, and 31.37% were located in the intron region (fig. S12D).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Replicates of four groups were clearly clustered separately.

“Replicates of four groups were clearly clustered separately as shown in fig. S12A.”

May combine two claims.

For WT-tFCI versus WT Sham, 27.74% of the open DARs in WT-tFCI microglia were located in the promoter region within 3 kb upstream of the TSS and 36.15% in the intron region, while 42.41% of the closed DARs were located in the promoter region and 28.81% in the intron region.

“Specifically, for WT-tFCI versus WT Sham, 27.74% of the open DARs in WT-tFCI microglia were located in the promoter region within 3 kb upstream of the transcription start site (TSS) region and 36.15% in the intron region, while 42.41% of the closed DARs were located in the promoter region and 28.81% in the intron region (fig. S12C).”

Probably not a substantial claim: may not pass S3 (load-bearing).

PCA interpreted large variations among the four groups.

“PCA interpreted large variations among the four groups (Fig. 7A, top left).”

May combine two claims.

Little overlap was observed between open DARs and up-regulated genes in miKO-tFCI versus WT-tFCI, but more than a half of the down-regulated genes had their corresponding promoters less accessible.

“Although little overlap was observed between open DARs and up-regulated genes in miKO-tFCI versus WT-tFCI, more than a half of the down-regulated genes had their corresponding promoters less accessible (fig. S12G).”

May combine two claims.

Although transcriptomically HDAC3-miKO-induced up-regulated gene counts are comparable to the down-regulated gene counts, ATAC-seq analysis indicated that HDAC3 deletion results in much more down-regulated loci than up-regulated loci.

“Although transcriptomically HDAC3-miKO–induced up-regulated gene counts are comparable to the down-regulated gene counts, ATAC-seq analysis indicated that HDAC3 deletion results in much more down-regulated loci than up-regulated loci.”

May combine two claims.

In the absence of HDAC3, the blockade of proliferation did not occur in Arg1+ or CD206+ microglia, while CD16+ microglia displayed comparable proliferative suppression as in the whole microglia population.

“Our results showed that in the absence of HDAC3, the blockade of proliferation did not occur in Arg1+ or CD206+ microglia, while CD16+ microglia displayed comparable proliferative suppression as in the whole microglia population.”

• AssumptionRests on an assumption: CD16 labels proinflammatory microglia and Arg1 labels anti-inflammatory microglia.
• AssumptionRests on an assumption: CD206 labels anti-inflammatory microglia/macrophage.

May combine two claims.

HDAC3 deficiency alleviated neuroinflammation and reduced the number of CD16+ microglia.

“Consistently, we also observed that HDAC3 deficiency alleviated neuroinflammation and reduced the number of CD16+ microglia.”

• AssumptionRests on an assumption: CD16 labels proinflammatory microglia and Arg1 labels anti-inflammatory microglia.

May combine two claims.

The HDAC3/PU.1 axis mediates differential proliferation-related reprogramming in different microglia populations, drives poststroke inflammatory state transition, and contributes to the pathophysiology of ischemic stroke.

“These findings uncovered that HDAC3/PU.1 axis, which mediated differential proliferation-related reprogramming in different microglia populations, drove poststroke inflammatory state transition, and contributed to pathophysiology of ischemic stroke.”

May combine two claims.

HDAC3 deficiency induces G2-M arrest and S-phase arrest in microglia after tFCI.

“suggesting induction of G2-M arrest and S-phase arrest.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Early microgliogenesis is dependent on PU.1 level.

“Early microgliogenesis is dependent on PU.1 level (13).”

• cites [13] K. Kierdorf, D. Erny, T. Goldmann, V. Sander, C. Schulz, E. G. Perdiguero, P. Wieghofer, A. Heinrich, P. Riemke, C. Hölscher, D. N. Müller, B. Luckow, T. Brocker, K. Debowski, G. Fritz, G. Opdenakker, A. Diefenbach, K. Biber, M. Heikenwalder, F. Geissmann, F. Rosenbauer, M. Prinz, Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat. Neurosci. 16, 273– 280 (2013). 23334579 sourceNot checked — no full text of the source

May combine two claims.

After the onset of stroke, microglia developed into different inflammation-associated phenotypes, which simultaneously underwent proliferation that was regulated by HDAC3 in a phenotype-dependent manner, eventually resulting in altered microglial states in miKO-tFCI mice.

“Therefore, we reasoned that after the onset of stroke, microglia developed into different inflammation-associated phenotypes, which simultaneously underwent proliferation that was regulated by HDAC3 in a phenotype-dependent manner, eventually resulting in altered microglial states in miKO-tFCI mice.”

May combine two claims.

The absence of HDAC3 efficiently ameliorated the peri-infarct inflammatory microenvironment due to the inhibited proliferation of proinflammatory microglia.

“suggesting that the absence of HDAC3 efficiently ameliorated the peri-infarct inflammatory microenvironment due to the inhibited proliferation of proinflammatory microglia.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The large variability in the percentage of DARs in the promoter region may indicate strong surgery effects and genotype effects.

“The large variability in the percentage of DARs in the promoter region may indicate strong surgery effects and genotype effects.”

May combine two claims.

The mitotic phase is the most critical and most variable process subjected to HDAC3 expression in the context of ischemic stroke.

“suggesting that the mitotic phase was the most critical as well as the most variable process subjected to HDAC3 expression in the context of ischemic stroke.”

May combine two claims.

These mitosis terms were similar to previous RNA-seq data, suggesting epigenetically regulated proliferation in miKO-tFCI microglia.

“which were similar to our previous RNA-seq data (Fig. 3, F and G), suggesting epigenetically regulated proliferation in miKO-tFCI microglia.”

May combine two claims.

Ablation of HDAC3 in microglia suppresses the expression of M phase-related genes and thus microglial proliferation.

“our data provided preliminary evidence that ablation of HDAC3 in microglia suppressed the expression of M phase–related genes and thus microglial proliferation.”

May combine two claims.

FACS isolation and qRT-PCR verify the deletion of HDAC3 in microglia.

“To verify the efficiency of deleting microglial HDAC3, on day 30 after tamoxifen administration, we used fluorescence-activated cell sorting (FACS) to isolate cerebral microglia”

• Also stated in: § Microglial HDAC3 expression increases after ischemia and the engineering of HDAC3-miKO mice · plain

  “quantification of Hdac3 mRNA expression in these FACS-isolated microglia revealed that Hdac3 was almost eliminated in microglia”

May combine two claims.

The consistency excluded the possibility that contamination of Arg1+ myeloid cells or CD206+ BAMs masked the suppressed proliferative potential of Arg1+ or CD206+ HDAC3-deficient microglia and validated that HDAC3 deficiency-induced proliferative effects had cell-specific preference.

“This consistency did exclude the possibility that contamination of Arg1+ myeloid cells or CD206+ border-associated macrophages (BAMs) masked the suppressed proliferative potential of Arg1+ or CD206+ HDAC3-deficient microglia and further validated that HDAC3 deficiency–induced proliferative effects had cell-specific preference.”

May combine two claims.

This suggests its implication in HDAC3-mediated transcriptional regulation, validated by the consistent evidence that inhibiting HDAC3 down-regulated microglial PU.1 in both RNA and protein levels.

“suggesting its implication in HDAC3-mediated transcriptional regulation, which is validated by the consistent evidence that inhibiting HDAC3 down-regulated microglial PU.1 in both RNA and protein levels.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The down-regulation of PU.1 may also involve RUNX1, which is a component of the HDAC3 corepressor complex and has been shown to bind to crucial sites within the PU.1 upstream regulatory element and regulate PU.1 expression.

“In addition, the down-regulation of PU.1 may also involve RUNX1, which is a component of the HDAC3 corepressor complex and has been shown to bind to crucial sites within the PU.1 upstream regulatory element and regulate PU.1 expression (53).”

• cites [53] G. Huang, P. Zhang, H. Hirai, S. Elf, X. Yan, Z. Chen, S. Koschmieder, Y. Okuno, T. Dayaram, J. D. Growney, R. A. Shivdasani, D. G. Gilliland, N. A. Speck, S. D. Nimer, D. G. Tenen, PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis. Nat. Genet. 40, 51– 60 (2008). 17994017 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

HDAC3 has been reported to regulate the progression of different cell cycle phases in different cells.

“During the past few years, HDAC3 has been reported to regulate the progression of different cell cycle phases in different cells (44).”

• cites [44] R. Sarkar, S. Banerjee, S. A. Amin, N. Adhikari, T. Jha, Histone deacetylase 3 (HDAC3) inhibitors as anticancer agents: A review. Eur. J. Med. Chem. 192, 112171 (2020). 32163814 sourceNot checked — no full text of the source

May combine two claims.

PU.1 is involved as a downstream molecule of HDAC3 in the selective inhibition of microglial proliferation and in the protection effects of HDAC3-miKO after tFCI.

“They provided strong support for the involvement of PU.1 as a downstream molecule of HDAC3 in the selective inhibition of microglial proliferation and, thereby, in the protection effects of HDAC3-miKO after tFCI.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Surgeries were performed by investigators blinded to animal genotypes and experimental grouping.

“Surgeries were performed by investigators blinded to animal genotypes and experimental grouping.”

Probably not a substantial claim: may not pass S2 (checkable).

This indicates a need for further exploration of the presence and role of CD206 on microglia in various diseases.

“indicating a need for further exploration of the presence and role of CD206 on microglia in various diseases (fig. S10D).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All animal experiments were approved by the Animal Care and Use Committee of Fudan University and reported following the ARRIVE guidelines.

“All animal experiments were approved by the Animal Care and Use Committee of Fudan University (approval numbers 20200306-117 and 20230-004S) and reported following the ARRIVE guidelines (75).”

• cites [75] C. Kilkenny, D. G. Altman, Improving bioscience research reporting: ARRIVE-ing at a solution. Lab. Anim 44, 377– 378 (2010). 20660161 sourceNot checked — no full text of the source

May combine two claims.

These microglia in two tFCI groups also undergo the similar so-called repopulation, which may confer different characteristics in turn to these renewed microglia.

“Regardless of the extent, these microglia in two tFCI groups also undergo the similar so-called repopulation, which may confer different characteristics in turn to these renewed microglia (63).”

• cites [63] E. F. Willis, K. P. A. MacDonald, Q. H. Nguyen, A. L. Garrido, E. R. Gillespie, S. B. R. Harley, P. F. Bartlett, W. A. Schroder, A. G. Yates, D. C. Anthony, S. Rose-John, M. J. Ruitenberg, J. Vukovic, Repopulating microglia promote brain repair in an IL-6-dependent manner. Cell 180, 833– 846.e16 (2020). 32142677 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Differential expression analysis was performed with batch differences controlled.

“Differential expression analysis was performed with batch differences controlled.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Inbuilt DEseq2 was invoked to perform the normalization and differential analysis, with batch effect considered.

“Inbuilt DEseq2 was then invoked to perform the normalization and differential analysis, with batch effect considered.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For integrative analysis of bulk RNA-seq and ATAC-seq, a permutation test was performed to check whether closed DARs overlap with down-regulated DEGs more than expected.

“To check whether closed DARs overlap with down-regulated DEGs more than expected, a permutation test was performed with an R package regioneR by the “permTest” function (ntimes = 200, randomize.function = randomizeRegions, evaluate.function = numOverlaps, universe = all.genes); all consensus peaks were assigned to the parameter, “all.genes.””

Classified as probably not a substantial claim.

Mice with a drop of less than 75% in cerebral blood flow compared to pre-ischemia levels were eliminated from the tFCI experiments.

“To ensure the success of the tFCI model, the blood flow of the middle cerebral artery branches (rCBF) was monitored by laser Doppler flowmetry (PeriFlux System 5001, Perimed, Jarfalla, Sweden) and laser speckle (PeriCam PSI System, Perimed, Jarfalla, Sweden). The mice with a drop of less than 75% in cerebral blood flow compared to pre-ischemia levels would be eliminated.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The volume of brain tissue loss was quantified by a researcher blinded to group allocation.

“The volume of brain tissue loss was quantified by a researcher blinded to group allocation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The normalized rlog/vst-transformed counts were adjusted using the removeBatchEffect from the limma package.

“The normalized rlog/vst-transformed counts were adjusted using the removeBatchEffect from the limma package.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Male mice were used for most experiments; female mice were exclusively used for behavioral testing and histological evaluation; all remaining trials were conducted exclusively on male mice for consistency.

“Of note, male mice were used for most experiments. Female mice were exclusively used for behavioral testing and histological evaluation (i.e., NeuN assessment) to confirm the presence of protective effects similar to those observed in male mice. Apart from these experiments, all the remaining trials were conducted exclusively on male mice for consistency.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All behavioral tests were performed by an investigator blinded to experimental manipulations.

“All behavioral tests were performed by an investigator blinded to experimental manipulations (detailed in Supplementary Materials and Methods).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All efforts were made to minimize animal suffering and the number of animals used.

“All efforts were made to minimize animal suffering and the number of animals used.”