TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation

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

ICE has been shown to interact with the Ifnb1 promoter in human fibroblasts in the absence of any viral infection.

“ICE has been recently shown to interact with the Ifnb1 promoter in human fibroblasts in the absence of any viral infection22.”

• cites [22] Banerjee, A. R., Kim, Y. J. & Kim, T. H. A novel virus-inducible enhancer of the interferon-beta gene with tightly linked promoter and enhancer activities. Nucleic Acids Res. 42, 12537–12554 (2014) . sourceSupported

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

Several TRIM proteins regulate production and action of IFN-β during innate immune response.

“Several TRIM proteins regulate production and action of IFN-β during innate immune response.”

• cites [34] Rajsbaum, R. et al. Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKepsilon kinase-mediated antiviral response. Immunity 40, 880–895 (2014) . sourceSupported
• cites [35] Inn, K. S. et al. Linear ubiquitin assembly complex negatively regulates RIG-I- and TRIM25-mediated type I interferon induction. Mol. Cell 41, 354–365 (2011) . sourcePartly supported

  The Results section ('HOIL-1L/HOIP complex negatively regulates RIG-I-mediated type-I IFN induction'; page unavailable) shows that TRIM25 promotes RIG-I-mediated IFN-β promoter activation. The paper supports a role for TRIM25 in IFN-β production, but does not establish that several TRIM proteins regulate it or that they regulate IFN-β’s action. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [36] Higgs, R. et al. The E3 ubiquitin ligase Ro52 negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. J. Immunol. 181, 1780–1786 (2008) . sourcePartly supported

  The Abstract (page unavailable) reports that Ro52/TRIM21 limits IFN-β production by promoting IRF3 degradation. The Introduction (page unavailable) also identifies TRIM5α and TRIM25 as important in antiviral defense, but does not establish that several TRIM proteins regulate IFN-β production or its action specifically. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

PU.1 can interact with CBP/p300 and TRIM33.

“as PU.1 can interact with CBP/p300 (refs 39, 40) and TRIM33”

• cites [39] Yamamoto, H., Kihara-Negishi, F., Yamada, T., Hashimoto, Y. & Oikawa, T. Physical and functional interactions between the transcription factor PU.1 and the coactivator CBP. Oncogene 18, 1495–1501 (1999) . sourceNot checked — no full text of the source
• cites [40] Bai, Y., Srinivasan, L., Perkins, L. & Atchison, M. L. Protein acetylation regulates both PU.1 transactivation and Ig kappa 3' enhancer activity. J. Immunol. 175, 5160–5169 (2005) . sourceSupported

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

Such enhancer or repressor loop stability has been described during development where they are associated with paused polymerase and in human fibroblasts where TNF-α-responsive enhancers contact their target promoters before TNF-α activation.

“Such enhancer or repressor loop stability has been described during development where they are associated with paused polymerase28 and in human fibroblasts where TNF-α-responsive enhancers contact their target promoters before TNF-α activation29.”

• cites [28] Ghavi-Helm, Y. et al. Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512, 96–100 (2014) . sourceNot checked — no full text of the source
• cites [29] Jin, F. et al. A high-resolution map of the three-dimensional chromatin interactome in human cells. Nature 503, 290–294 (2013) . sourceSupported

May combine two claims.

TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts.

“Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts.”

• Also stated in: § TRIM33 controls Ifnb1 expression in activated macrophages · plain

  “Altogether, these results show that TRIM33 deficiency is associated with increased Ifnb1 mRNA level associated with IFN-β secretion during the late stages of LPS activation of BMDM.”

• Also stated in: § TRIM33 controls Ifnb1 expression in activated macrophages · hedged

  “Finally, highly efficient shRNA-mediated depletion of TRIM33 in NIH3T3 cells (NIH3T3 TRIM33−; Supplementary Fig. 1e) did not modify kinetics of Ifnb1 mRNA levels in response to poly(I:C) (Fig. 1f), indicating cell-specificity in the regulation of Ifnb1 expression by TRIM33.”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.
• AssumptionRests on an assumption: The LyzCre-mediated deletion of Trim33 in macrophages does not cause developmental or compensatory changes that confound the Ifnb1 phenotype.

May combine two claims.

In LPS-activated WT and Trim33−/− iM, Ifnb1 mRNA increases 1 h after activation and peaks at 2 h, with twofold higher expression in WT iM than in Trim33−/− iM.

“Ifnb1 mRNA levels increased 1 h after LPS activation of WT and Trim33−/− iM and peaked at 2 h with twofold higher observed expression in WT iM than in Trim33−/− iM”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.

May combine two claims.

These epigenetic modifications are present in ST-HSCs and myeloid cells, but not in mature erythroid and lymphoid cells.

“These epigenetic modifications are present in short-term haematopoietic stem cells (ST-HSCs) and in myeloid cells, but not in mature erythroid and lymphoid cells”

• cites ref. 24Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

In WT BMDM and PM activated by LPS, Ifnb1 mRNA levels decrease from 2 h (BMDM) or 4 h (PM) and return to basal by 6 h, whereas in Trim33−/− BMDM and PM they rise significantly by 6 h and remain high 24 h after LPS.

“Thereafter, Ifnb1 mRNA levels decreased from 2 h in WT BMDM (or 4 h in WT PM) and returned to basal levels by 6 h in WT BMDM and PM. In contrast, although Ifnb1 mRNA levels decreased between 2 and 4 h, Ifnb1 mRNA levels began to rise significantly by 6 h and remained high 24 h after LPS activation in Trim33−/− BMDM or PM”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.

May combine two claims.

RAW 264.7 clones with biallelic deletion of ICE show high and sustained Ifnb1 mRNA levels after LPS or poly(I:C) activation, with highest increase during late phase.

“In the two clones high and sustained Ifnb1 mRNA levels were found but only after LPS or poly(I:C) activation, with the highest increase being obtained during the late phase of activation”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.
• AssumptionRests on an assumption: The CRISPR/Cas9-mediated deletion of ICE does not affect other regulatory elements or gene function beyond the intended deletion.

May combine two claims.

TRIM33 deficiency increases Ifnb1 mRNA levels in NIH3T3 PU.1+ cells treated with poly(I:C) but does not result in sustained expression during late stages.

“TRIM33 deficiency increased Ifnb1 mRNA levels in NIH3T3 PU.1+ cells treated with poly(I:C) but did not result in sustained expression of Ifnb1 during the late stages of poly(I:C) activation”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.

May combine two claims.

Late sustained Ifnb1 expression is also observed with poly(I:C) in BMDM and PM, with much higher Ifnb1 mRNA at all late time points in poly(I:C)-treated Trim33−/− BMDM compared with WT.

“This late sustained expression of Ifnb1 was also observed when BMDM and PM were stimulated by poly(I:C) with Ifnb1 mRNA levels being much higher at all late time points in poly(I:C) treated Trim33−/− BMDM compared with WT”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.

May combine two claims.

In WT iM, Ifnb1 mRNA levels drop after peak, whereas in Trim33−/− iM they remain high.

“As anticipated, Ifnb1 mRNA levels in WT iM then dropped, whereas in Trim33−/− iM, Ifnb1 mRNA levels remained high”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.

May combine two claims.

Trim33 deletion occurs in monocytes, bone-marrow-derived macrophages, peritoneal macrophages and neutrophils but not in myeloid progenitors or B lymphocytes.

“PCR genotyping of purified haematopoietic cell populations showed deletion of Trim33 in monocytes, bone-marrow-derived macrophages (BMDM), peritoneal macrophages (PMs) and neutrophils but not in myeloid progenitors, nor in B lymphocytes”

May combine two claims.

RNA Polymerase II and H3K4me3 are constitutively bound at high levels on ICE.

“ChIP and ChIP-seq analyses showed a constitutive binding of RNA Polymerase II (Pol II) and constitutively high levels of H3K4me3 on ICE”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

PU.1 binding at the Ifnb1 promoter and at ICE is constitutive and not TRIM33-dependent.

“PU.1 binding at the Ifnb1 promoter and at ICE was constitutive and detected in WT and Trim33−/− BMDM (Fig. 2b, middle and right panels) indicating that PU.1 binding to ICE was not TRIM33-dependent.”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

TRIM33 mutants lacking ubiquitin ligase activity or Smad-interaction domain rescue normal Ifnb1 expression at late time points in LPS-activated Trim33−/− iM, whereas a mutant lacking the coiled-coil domain does not.

“The flag-ΔUb and the flag-ΔSmad rescued normal Ifnb1 expression at late time points of LPS-activated Trim33−/− iM whereas the flag-ΔCC did not rescue normal profile of Ifnb1 expression in activated Trim33−/− iM”

May combine two claims.

No TRIM33 binding is observed on the Ifnb1 promoter or ICE in NIH3T3 cells, indicating macrophage specificity of TRIM33 binding to ICE.

“Finally, no TRIM33 binding was observed on the Ifnb1 promoter or ICE in NIH3T3 cells (Fig. 2c), indicating macrophage specificity of TRIM33 in binding to ICE.”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

Human and mouse ICE sequences show 70% homology around the TRIM33/PU.1 peak with consensus binding motifs for AP-1, NF-κB and IRF transcription factors.

“Comparison of reported human and mouse ICE sequences showed 70% sequence homology around the TRIM33/PU.1 peak with consensus binding motifs for AP-1, NF-κB and IRF transcription factors”

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

Trim33−/− mice did not display developmental abnormalities and were healthy.

“Trim33−/− mice did not display developmental abnormalities and were healthy.”

May combine two claims.

In LPS-activated RAW ICE+/− cells, Ifnb1 mRNA levels increase comparably to WT but subsequently remain stable, whereas in WT they gradually decrease.

“In LPS-activated RAW WT cells, Ifnb1 mRNA levels increased during the first 4 h and then gradually decreased (Supplementary Fig. 2e). In contrast, whereas Ifnb1 mRNA levels increased comparably in RAW ICE+/− cells, they subsequently remained stable”

• AssumptionRests on an assumption: The Ifnb1 mRNA levels measured reflect Ifnb1 gene transcription.
• AssumptionRests on an assumption: The CRISPR/Cas9-mediated deletion of ICE does not affect other regulatory elements or gene function beyond the intended deletion.

May combine two claims.

Prior to LPS activation, H3ac on ICE is similar in WT and Trim33−/− BMDM; during LPS activation, H3ac on ICE does not change in WT but transiently increases 10 h after LPS in Trim33−/− BMDM.

“Prior to LPS activation, histone H3 acetylation (H3ac) on ICE was similar in WT and Trim33−/− BMDM (Fig. 3c, left panel). During LPS activation, H3ac on ICE did not change in WT BMDM but was transiently increased 10 h after LPS activation in Trim33−/− BMDM”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

In WT BMDM, 3C-seq shows a loop between ICE and the Ifnb1 gene before LPS activation and a twofold stronger interaction 24 h after LPS activation.

“In WT BMDM, both viewpoints showed a loop between ICE and the Ifnb1 gene before LPS activation and a twofold stronger interaction 24 h after LPS activation”

• AssumptionRests on an assumption: The 3C-seq signals reflect physical chromatin loops between ICE and Ifnb1.

May combine two claims.

TRIM33 ChIP-seq in RAW 264.7 or LPS-treated BMDM shows a single TRIM33 peak at the Ifnb1 locus overlapping a PU.1 peak 15 kb upstream of the Ifnb1 TSS.

“TRIM33 ChIP-seq data obtained from the RAW 264.7 murine macrophage cell line or from BMDM treated with LPS for 24 h showed a single TRIM33 peak at the Ifnb1 locus that overlapped with a PU.1 peak located 15 kb upstream from the Ifnb1 TSS”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

TRIM33 inactivation leads to recruitment of CBP/p300 at the Ifnb1 promoter and enhanced H3ac.

“First, TRIM33 inactivation led to recruitment of CBP/p300 at the Ifnb1 promoter and enhanced H3ac.”

May combine two claims.

In WT BMDM, Pol II occupancy at TSS and in Ifnb1 3′ region increases after 1 h of LPS and is lost at 4 h; in Trim33−/− BMDM, Pol II occupancy increases after 1 h but is maintained up to 24 h.

“In WT BMDM, Pol II occupancy at TSS and in Ifnb1 3′ region increased after 1 h of stimulation and then was lost at 4 h (Fig. 4b, upper panels). In Trim33−/− BMDM, Pol II occupancy similarly increased after 1 h of stimulation but its levels were maintained up to 24 h”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

H3K4me3 levels are transiently increased in WT BMDM after 4 h of LPS, whereas in Trim33−/− BMDM persistent and high levels are observed up to 24 h.

“H3K4me3 levels were transiently increased in WT BMDM after 4 h of LPS stimulation, whereas in Trim33−/− BMDM, persistent and high levels of H3K4me3 were also observed up to 24 h”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

Adding C646, a CBP/p300 inhibitor, 2 h after LPS induction does not modify Ifnb1 expression in WT BMDM but reverses the high sustained Ifnb1 transcription level in Trim33−/− BMDM at both 10 and 24 h.

“Adding C646 2 h after LPS induction did not modify Ifnb1 expression in WT BMDM, but reversed the high sustained Ifnb1 transcription level in Trim33−/− BMDMs at both 10 and 24 h”

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

PU.1 is not expressed in fibroblasts.

“Indeed, PU.1, which is not expressed in fibroblasts, is bound to ICE in macrophages and might be involved in the cell-specific function of ICE.”

May combine two claims.

In WT BMDM, c-jun and p65 are recruited to the Ifnb1 promoter at 1 h after LPS activation and their binding returns to basal levels after 24 h.

“As anticipated, in WT BMDM, c-jun and p65 were recruited to the Ifnb1 promoter at early times following LPS activation (1 h), but their binding returned to basal levels after 24 h”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

Expression of ectopic full-length flag-TRIM33 in Trim33−/− iM decreases H3ac on the Ifnb1 promoter 7 h after LPS stimulation, whereas flag-ΔCC does not.

“Expression of ectopic full-length flag-TRIM33 in Trim33−/− iM led to decreased H3ac on the Ifnb1 promoter 7 h after LPS stimulation (Fig. 4d), whereas the flag-ΔCC that could not rescue normal Ifnb1 expression in Trim33−/− iM (Fig. 1h) did not decrease H3ac on the Ifnb1 promoter 7 h after LPS stimulation”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

Prior to LPS activation, histone H3ac on the Ifnb1 promoter is similar in WT and Trim33−/− BMDM; it increases at 1 h in both, returns to basal in WT, but shows a 5- to 8-fold increase at 10 and 24 h in Trim33−/− BMDM.

“Prior to LPS activation, histone H3ac on the Ifnb1 promoter was similar in WT and Trim33−/− BMDM (Fig. 4c). This H3ac increased at 1 h after LPS activation of WT and Trim33−/− BMDM. It then returned to basal levels in WT BMDM, whereas a 5- to 8-fold increase in H3ac was observed after 10 and 24 h in Trim33−/− BMDM”

• AssumptionRests on an assumption: The ChIP and ChIP-seq signals for TRIM33, PU.1, Pol II, histone modifications and CBP/p300 are specific to their intended targets.

May combine two claims.

Treatment of LPS-activated WT BMDM with trichostatine A, an HDAC inhibitor, does not result in sustained Ifnb1 expression, whereas it alters expression of genes regulated by HDACs.

“Treatment of LPS-activated WT BMDM with trichostatine A, an inhibitor of HDACs, did not result in sustained Ifnb1 expression, whereas it did alter expression of genes regulated by HDACs”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

In Trim33−/− BMDM, p65 is loaded onto the enhanceosome during the late phases of LPS activation.

“This TRIM33-dependent recruitment and activity of CBP/p300 could be direct and/or mediated by trans-acting factors such as p65 that are, in Trim33−/− BMDM, loaded onto the enhanceosome during the late phases of LPS activation.”

May combine two claims.

The data describe a new regulatory layer through identification of cis- and trans-acting elements that regulate Ifnb1 gene transcription at specific steps of macrophage activation, but not in fibroblasts.

“Our data describe a new regulatory layer through identification of cis- and trans-acting elements that regulate Ifnb1 gene transcription at specific steps of macrophage activation, but not in fibroblasts.”

May combine two claims.

TRIM33 deficiency does not modify ICE chromatin signature and only leads to transient increases of H3ac and CBP/p300 recruitment on ICE, whereas sustained high Ifnb1 expression is observed, suggesting TRIM33 bound to ICE might act at a distance.

“TRIM33 deficiency did not modify ICE chromatin signature and only led to transient increases of H3ac and CBP/p300 recruitment on ICE whereas a sustained high expression of Ifnb1 was observed in LPS-activated Trim33−/− BMDM suggesting that TRIM33 bound to ICE might act at a distance.”

May combine two claims.

TRIM33 can repress Ifnb1 expression in the presence of PU.1, and additional factors not present in NIH3T3 cells are required for TRIM33-mediated late downregulation of Ifnb1 expression.

“These results show that TRIM33 can repress Ifnb1 expression in presence of PU.1 and indicate that additional factors that are not present in NIH3T3 cells are required for TRIM33-mediated late downregulation of Ifnb1 expression.”

May combine two claims.

ICE functions as a cis-acting transcriptional repressor element of Ifnb1 gene activation in macrophages, and TRIM33 and PU.1 bound to ICE may have an important role in Ifnb1 gene transcription shutdown during late phase of macrophage activation.

“Altogether, these results show that ICE functions as a cis-acting transcriptional repressor element of the Ifnb1 gene activation in macrophages and indicate that TRIM33 and PU.1 bound to ICE may have an important role in Ifnb1 gene transcription shutdown during the late phase of macrophage activation.”

May combine two claims.

The Smad-interaction and ubiquitin ligase domains are not necessary for TRIM33 regulation of Ifnb1 expression, suggesting TRIM33 is part of a regulatory complex that regulates Ifnb1 transcription.

“These results indicate that the Smad-interaction and ubiquitin ligase domains are not necessary for TRIM33 regulation of Ifnb1 expression and suggest that TRIM33 is part of a regulatory complex that regulates Ifnb1 transcription.”

Classified as probably not a substantial claim.

Firefly luciferase activity was normalized with respect to renilla luciferase activity.

“Firefly luciferase activity was normalized with respect to renilla luciferase activity.”

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

The opposite roles of ICE in fibroblasts and macrophages might be linked to recruitment of distinct proteins in the two cell types.

“These opposite roles of ICE in fibroblasts and macrophages might be linked to recruitment of distinct proteins in the two cell types.”

May combine two claims.

The activity and/or substrate availability of TRIM33 regulates Ifnb1 expression at the end of LPS activation of macrophages.

“Together with the continuous binding of TRIM33 on ICE, the complementation of the Ifnb1 phenotype in macrophages by re-expression of TRIM33 in Trim33−/− immortalized macrophages suggested a direct role of TRIM33 in Ifnb1 transcription and indicated that the activity and/or substrate availability of TRIM33 regulated Ifnb1 expression at the end of LPS activation of macrophages.”

Classified as probably not a substantial claim.

All the clones were verified by sequencing of a 2 kb genomic region containing the ICE.

“All the clones were verified by sequencing of a 2 kb genomic region containing the ICE.”

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

All the constructs were verified by sequencing.

“All the constructs were verified by sequencing.”

May combine two claims.

In WT BMDM, Pol II drops off and Ifnb1 transcription ceases, whereas in Trim33−/− BMDM, continued Pol II occupancy and H3K4me3 promote sustained Ifnb1 transcription.

“Then, in WT BMDM, Pol II drops off and Ifnb1 transcription ceases, whereas in Trim33−/− BMDM, continued Pol II occupancy and H3K4me3 promote sustained Ifnb1 transcription.”

May combine two claims.

The ICE/Ifnb1 loop is cell-type independent, stable and independent of viral or bacterial stimulation.

“These combined findings indicate that ICE/Ifnb1 loop is cell-type independent, stable and independent of viral or bacterial stimulation.”

May combine two claims.

ICE has an open-chromatin structure highly conserved during myeloid differentiation and characterized by Pol II occupancy and H3K4 trimethylation, features not typical of an enhancer or repressor.

“ICE has an open-chromatin structure highly conserved during myeloid differentiation and characterized by Pol II occupancy and tri-methylation of H3K4, two features that are not typical marks of an enhancer or a repressor.”

May combine two claims.

ICE interacts with the Ifnb1 proximal region in a constitutive and TRIM33-independent manner, but this interaction is strengthened following LPS stimulation.

“These results indicate that ICE interacts with the Ifnb1 proximal region in a constitutive and TRIM33-independent manner, but that this interaction is strengthened following LPS stimulation.”

May combine two claims.

Enhanced CBP/p300 recruitment and activity at late times of activation is required for sustained Ifnb1 expression in Trim33−/− BMDM, and TRIM33 may regulate Ifnb1 expression at late times by preventing the recruitment of CBP/p300.

“Altogether, these results show that enhanced CBP/p300 recruitment and activity at late times of activation is required for sustained Ifnb1 expression in Trim33−/− BMDM and that TRIM33 may regulate Ifnb1 expression at late times by preventing the recruitment of CBP/p300.”

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

All adherent cells expressed F4/80 and were considered as PMs.

“All adherent cells expressed F4/80 and were considered as PMs.”

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

gRNA were designed to minimize the number of off-target sites.

“Two guides RNA (gRNA) were designed to delete a 1,361 bp fragment containing the −15 kb region using the CRISPR Design Tool (http://tools.genome-engineering.org), to minimize the number of off-target sites.”

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

Single clones were isolated that expressed F4/80, CD11b but not Gr1.

“Single clones were isolated that expressed F4/80, CD11b but not Gr1.”

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

Experiments were performed in compliance with European legislation and with the Ethics Committee of the French Ministry of Agriculture.

“Experiments were performed in compliance with European legislation and with the Ethics Committee of the French Ministry of Agriculture (Agreement B9203202).”