Oxylipin biosynthesis reinforces cellular senescence and allows detection of senolysis

May combine two claims.

CAY and NS decrease CDKN1A/p21 and CDKN2A/p16INK4a mRNA levels.

“and decreased both CDKN1A (p21) and CDKN2A (p16INK4a) mRNA levels (Figures 4D and 4E).”

May combine two claims.

Both compounds reduce secreted IL-6 levels in SEN(IR) cells.

“Both compounds reduced secreted IL-6 levels (Figure 4F).”

May combine two claims.

Both lipids induce several SASP factor mRNAs, and dihomo-15d-PGJ2 induces higher levels than 15d-PGJ2.

“Further, although both lipids induced several SASP factor mRNAs, dihomo-15d-PGJ2 induced higher levels of these mRNAs than 15d-PGJ2 (Figure 4N).”

May combine two claims.

One shRNA reduces PTGDS expression and also lowers several SASP factor mRNAs compared with control shRNA.

“One shRNA efficiently reduced PTGDS expression (Figure S4P), and also reduced several SASP factor mRNAs compared to senescent cells transduced with a control shRNA (shGFP) (Figure 4O).”

May combine two claims.

PGDH mRNA decreases in replicatively senescent fibroblasts and IR-induced senescent HUVECs.

“Indeed, PDGH mRNA levels decreased in replicatively senescent fibroblasts and IR-induced senescent HUVECs (Figure 1J, Figure S1G).”

May combine two claims.

All senescent cell types tested secrete more PGD2, though the ELISA cannot separate dihomo-PGD2 from PGD2.

“All senescent cells showed elevated PGD2 secretion, although the ELISA does not distinguish dihomo-PGD2 from PGD2.”

May combine two claims.

COX-2 inhibition by either compound lowers mRNAs for all inflammatory cytokines assayed and MMP3, but does not affect VEGF.

“COX-2 inhibition by either compound decreased mRNA levels of all the inflammatory cytokines assayed, as well as MMP3, but had no effect on VEGF – part of a pro-angiogenic subset of SASP factors (Figure 4G).”

May combine two claims.

SLCO2A1 mRNA increases less during replicative senescence and in IR-induced senescent HUVECs.

“Smaller increases occurred during replicative senescence (Figure S1) and in IR-induced senescent HUVECs (Figure 1J).”

May combine two claims.

Both PTGS2 inhibitors CAY-10404 and NS-398 lower secreted PGD2 levels.

“Both inhibitors suppressed secreted PGD2 levels, as determined by ELISA (Figure 4A).”

May combine two claims.

PGD2 and PGJ2 have similar effects on senescence phenotypes, and PGE2 has similar effects to a lesser degree.

“PGD2 and PGJ2 had similar effects on senescence phenotypes (Figures S4G–S4L), as did PGE2 to a lesser degree, as reported (Martien et al., 2013).”

• cites Martien et al., 2013 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

SEN(IR) media contain little (dh)15d-PGJ2, but much more after ABT-263-induced senolysis.

“Similarly, SEN(IR) media contained only small amounts of (dh)15d-PGJ2, but contained much more after ABT-263-mediated senolysis (Figure 2A).”

May combine two claims.

PGE2-induced phenotypes are unlikely to be senescence because PGE2-treated cells become smaller and resume proliferation after PGE2 removal, unlike PGD2- or PGJ2-treated cells.

“Phenotypes induced by PGE2 were unlikely due to senescence, as cells treated with PGE2 - but not PGD2 or PGJ2 - became smaller and resumed proliferation once PGE2 was removed (Figures S4N–S4O).”

May combine two claims. The quote only partly states this claim.

CAY and NS decrease SA-Bgal positivity.

“decreased senescence-associated beta-galactosidase (SA-Bgal) positivity (Figure 4C)”

May combine two claims.

Senescent cells have higher DHA, DPA, and erucic acid levels, but not behenic acid.

“Further, senescent cells contained higher levels of the 22-carbon PUFAs docosahexanoic acid (DHA) and docosapentanoic acid (DPA) and the 22-carbon MUFA erucic acid, but not the 22-carbon saturated fatty acid behenic acid.”

May combine two claims.

15d-PGJ2 or dihomo-15d-PGJ2 increases CDKN1A/p21 mRNA levels.

“and CDKN1A/p21 mRNA levels (Figure 4K).”

May combine two claims.

15d-PGJ2 or dihomo-15d-PGJ2 reduces LMNB1 mRNA.

“and LMNB1 mRNA (Figure 4M)”

May combine two claims.

Media from MiDAS cells contain some (dh)15d-PGJ2, and ABT-263 increases it significantly.

“Media from MiDAS cells (which have a low basal level of apoptosis) showed some (dh)15d-PGJ2, but this increased significantly when the cells were treated with ABT-263 (Figure 2A).”

May combine two claims.

Dihomo-15d-PGJ2 builds up inside senescent cells and is not substantially secreted.

“Since dihomo-15d-PGJ2 accumulated inside senescent cells (Figure 1A) and was not substantially secreted (Figure 2A–D)”

May combine two claims.

SLCO2A1 mRNA rises more than 100-fold in IR- and mitochondrial dysfunction-induced senescent fibroblasts and more than 50-fold in RAS-induced senescent cells.

“SLCO2A1 mRNA levels increased >100-fold in human fibroblasts induced to senesce by IR and mitochondrial dysfunction (Figure 1H) and >50-fold in RAS-induced senescent cells (Figure 1I).”

May combine two claims.

SEN(IR) cells show increased RAS activation and phospho-ERK1/2, which CAY or NS attenuates.

“Consistent with elevated 15d-PGJ2 levels, SEN(IR) cells showed increased RAS activation (RAS:GTP) and phospho-ERK1/2, which was attenuated by CAY or NS (Figure 5A).”

May combine two claims.

The DP1 agonist raises cAMP, while the DP2 agonist lowers cAMP, confirming receptor activation.

“The DP1 agonist increased cAMP levels, while the DP2 agonist lowered cAMP levels (Figure S5A), confirming receptor activation (Hata and Breyer, 2004; Hirai et al., 2001; Hirata et al., 1994; Ricciotti and FitzGerald, 2011).”

• cites Hata and Breyer, 2004Not checked yet

  Not checked in the free run.

• cites Hirai et al., 2001Not checked yet

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• cites Hirata et al., 1994Not checked yet

  Not checked in the free run.

May combine two claims.

1 μM AZ 628 does not alter SA-b-gal activity or EdU labeling.

“Further, 1 μM AZ 628 did not alter SA-b-gal activity (Figure S5L–S5M) or EdU labeling (Figure S5N).”

May combine two claims.

Continuous AZ 628 prevents much of the rise in proinflammatory SASP factor mRNAs, lowers PTGS2 and PTGDS mRNAs, and antagonizes the positive feedback initiated by 15d-PGJ2.

“Finally, continuous culture of either SEN(IR) or 15d-PGJ2-treated cells with AZ 628 prevented much of the rise in proinflammatory SASP factor mRNAs, lowered levels of both PTGS2 and PTGDS mRNAs, and thus antagonized the positive feedback initiated by 15d-PGJ2 (Figure 5F).”

May combine two claims.

PGD2 induces many SASP factor mRNAs, but the DP1 agonist does not.

“PGD2 induced many mRNAs encoding SASP factors, but the DP1 agonist did not (Figure S5F).”

May combine two claims.

PGD2 raises p21 and p16INK4a mRNAs, the DP1 agonist lowers these mRNAs, and the DP2 agonist has no effect.

“PGD2 also increased mRNAs encoding p21 and p16INK4a (Figure S5C and D), whereas the DP1 agonist lowered these mRNA levels. The DP2 agonist had no effect.”

May combine two claims.

In cells induced to senesce by 15d-PGJ2, PRAK is phosphorylated on threonine 182, p53 is phosphorylated on serine 37, and p21 protein rises.

“Indeed, PRAK was phosphorylated on threonine 182 (PRAK-R182), p53 was phosphorylated on serine 37 and p21 protein levels increased in cells induced to senesce by 15d-PGJ2 (Figures 5C–5D).”

May combine two claims.

PPAR-γ reporter activity rises about 18-fold in senescence, and NS-398 lowers it by about 50%.

“PPAR-γ reporter activity increased ~18 fold upon senescence, and NS-398 lowered this activity by ~50% (Figure S5H)”

May combine two claims.

Both the DP1 agonist and PGD2 reduce EdU incorporation, but only PGD2 raises SA-Bgal activity.

“Both the DP1 agonist and PGD2 reduced EdU incorporation, but only PGD2 elevated SA-Bgal activity (Figure S5B).”

May combine two claims.

IR-induced senescence shows a similar loss of RRM2, which CAY or NS prevents.

“A similar loss of RRM2 occurred in IR-induced senescence, which was prevented by CAY or NS (Figure 5B).”

May combine two claims.

PTGDR mRNA is unchanged between senescent and non-senescent cells, while PTGDR2 mRNA is about 50-fold lower in senescent cells.

“Further, although PTGDR mRNA levels were unchanged between senescent and non-senescent cells, PTGDR2 mRNA was ~50-fold lower in senescent cells, and thus unlikely to affect senescence (Figure S5D).”

May combine two claims.

This accumulation is linked to increased prostaglandin transporter expression but not prostaglandin dehydrogenase expression.

“This accumulation was linked to increased expression of prostaglandin transporter, but not prostaglandin dehydrogenase”

May combine two claims.

Incubating quiescent cells with 15d-PGJ2 activates RAS and phosphorylates ERK1/2.

“Indeed, incubation of quiescent cells with 15d-PGJ2 resulted in RAS activation, as well as phosphorylation of the downstream effector kinases ERK1/2 (Figure 5A; left lanes) (Oliva et al., 2003).”

• cites Oliva et al., 2003Not checked yet

  Not checked in the free run.

May combine two claims.

RELA depletion does not affect prostaglandin synthase expression despite lowering IL6 expression.

“By comparison, depletion of the NF-κB subunit RELA using a lentiviral shRNA (shRELA) had no effect on prostaglandin synthase expression (Figures S5S–S5T), despite lowering IL6 expression (Figure S5U), as described (Freund et al., 2011).”

• cites Freund et al., 2011 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The PPAR-γ agonists ciglitazone and troglitazone do not induce senescence.

“Unlike 15d-PGJ2, neither agonist induced senescence, as determine by EdU incorporation and SA-Bgal activity (Figure S5I).”

May combine two claims.

AZ 628 prevents the rise in p21 and p16INK4a mRNAs and the reduction in LMNB1 mRNA after 10 Gy IR.

“AZ 628 also prevented the rise in mRNAs encoding p21 and p16INK4a and reduction in LMNB1 mRNA in response to 10 Gy IR (Figures S5O–S5Q).”

May combine two claims.

In response to 15d-PGJ2, AZ 628 blocks ERK1/2 and p53 phosphorylation and prevents the rise in p53 and p21 proteins.

“In response to 15d-PGJ2, AZ 628 blocked ERK1/2 and p53 phosphorylation, thus preventing the rise in p53 and p21 proteins (Figure 5D).”

May combine two claims.

AZ 628 strongly inhibits ERK phosphorylation after serum stimulation without changing basal p53 or p21.

“AZ 628 (20, 5 or 1 μM) strongly inhibited ERK phosphorylation in response to serum stimulation without affecting basal levels of p53 or p21 (Figure S5K).”

May combine two claims.

In senescent cells, the estimated mean intracellular concentration of dihomo-15d-PGJ2 is 1.8 μM by mass spectrometry or 5.8 μM by ELISA, based on a measured 69 nM in 1 mL of lysate from 5×10^6 cells and an assumed 7.5 pL cell volume.

“Based upon an average measured concentration of 69 nM in 1 mL of cell lysate representing 5 × 106 senescent cells, and assuming an average volume of 7.5 pL per senescent cell, we estimated a mean intracellular concentration of 1.8 μM by mass spectrometry, or 5.8 μM by ELISA.”

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

Senescent cells are known to make prostaglandin E2.

“Senescent cells are known to produce prostaglandin E2 (PGE2) (Cormenier et al., 2018; Dagouassat et al., 2013; Kabir et al., 2016; Martien et al., 2013; Yang et al., 2011; Zdanov et al., 2007)”

• cites Cormenier et al., 2018 sourceNot checked yet

  Not checked in the free run.

• cites Dagouassat et al., 2013 sourceNot checked yet

  Not checked in the free run.

• cites Kabir et al., 2016 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Senescent cells can cause age-related pathologies partly by releasing many biologically active molecules.

“Cell culture and transgenic mouse studies show that senescent cells can drive several age-associated pathologies in part by secreting a myriad of biologically active molecules.”

• cites Acosta et al., 2008 sourceNot checked yet

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• cites Coppe et al., 2008 sourceNot checked yet

  Not checked in the free run.

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

The number of senescent cells rises with age in human and mouse tissues.

“Senescent cells increase with age in human and mouse tissues (Campisi, 2013; Wiley and Campisi, 2016)”

• cites Campisi, 2013 sourceNot checked yet

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• cites Wiley and Campisi, 2016Not checked yet

  Not checked in the free run.

May combine two claims.

Prostaglandin synthesis promotes senescence-associated phenotypes, including cell cycle arrest and expression and secretion of certain SASP factors.

“Thus, prostaglandin synthesis promotes phenotypes associated with senescence, including cell cycle arrest and the expression and secretion of certain SASP factors.”

Classified as probably not a substantial claim.

Overexpression of cyclooxygenase 2 causes premature aging phenotypes in mice.

“we considered that cyclooxygenase 2 (Ptgs2) overexpression causes premature aging phenotypes in mice (Kim et al., 2016)”

• cites Kim et al., 2016 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

5-lipoxygenase activity rises in replicative and oncogene-induced senescence and strengthens senescence phenotypes by increasing reactive oxygen species.

“the leukotriene-producing 5-lipoxygenase activity was shown to increase in replicative and oncogene-induced senescence, where it reinforced senescence-associated phenotypes by increasing reactive oxygen species (Catalano et al., 2005).”

• cites Catalano et al., 2005Not checked yet

  Not checked in the free run.

May combine two claims.

Prostaglandin synthesis activates PPAR-γ in senescent cells, but PPAR-γ does not drive senescence phenotypes.

“Therefore, while prostaglandin synthesis indeed activates PPAR-γ in senescent cells, PPAR-γ does not drive senescence phenotypes.”

May combine two claims.

Exogenous 15d-PGJ2 activates H-RAS at 3-5 μM, close to the dihomo-15d-PGJ2 level in senescent cells.

“H-RAS is activated by exogenous 15d-PGJ2 at 3–5 μM (Oliva et al., 2003) – roughly the dihomo-15d-PGJ2 level in senescent cells”

• cites Oliva et al., 2003Not checked yet

  Not checked in the free run.

May combine two claims.

15d-PGJ2 is a PPAR-γ ligand, and PPAR-γ is linked to senescence.

“15d-PGJ2 is a ligand for PPAR-γ (Forman et al., 1995; Kliewer et al., 1995), which is linked to senescence (Gan et al., 2008).”

• cites Forman et al., 1995Not checked yet

  Not checked in the free run.

• cites Kliewer et al., 1995Not checked yet

  Not checked in the free run.

• cites Gan et al., 2008 sourceNot checked yet

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

Oncogenic RAS mutations are known to drive cells into senescence.

“While oncogenic RAS mutations are known to drive cells into senescence (Serrano et al., 1997)”

• cites Serrano et al., 1997 sourceNot checked yet

  Not checked in the free run.

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

Other cyclopentenone prostaglandins activate other RAS isoforms or similar GTPases.

“and other cyclopentenone prostaglandins activate other RAS isoforms or similar GTPases (Anta et al., 2016; Renedo et al., 2007; Wall et al., 2015).”

• cites Anta et al., 2016Not checked yet

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• cites Renedo et al., 2007Not checked yet

  Not checked in the free run.

• cites Wall et al., 2015Not checked yet

  Not checked in the free run.

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

Chorioamnion-derived PGE2 and PGF2α are key signals for parturition.

“For example, chorioamnion-derived PGE2 and PGF2α are key signals for parturition (Gibb, 1998)”

• cites Gibb, 1998Not checked yet

  Not checked in the free run.

May combine two claims.

The RAS-RAF signaling pathway is important for many aspects of senescence, including SASP components, higher p21 and p16INK4a, and lower lamin B1.

“We show that the RAS-RAF signaling pathway is important for many aspects of senescence, including components of the SASP, increased levels of p21 and p16INK4a, and decline in lamin B1 levels.”

Classified as probably not a substantial claim.

RAS-induced senescence is driven by a MAPK cascade that leads to p53 phosphorylation on serine 37 by PRAK/MAPKAPK5.

“RAS-induced senescence is driven by a mitogen activated kinase (MAPK) cascade, resulting in p53 phosphorylation on serine 37 (p53-S37) by p38-regulated activated kinase (PRAK/MAPKAPK5) (Sun et al., 2007).”

• cites Sun et al., 2007 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Prostaglandins contribute to, but are not exclusively required for, secretion of proinflammatory factors.

“suggesting that prostaglandins contribute to, but are not exclusively necessary for, the secretion of proinflammatory factors.”

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

15d-PGJ2 binds preferentially to DP2.

“15d-PGJ2 preferentially binds DP2 (Monneret et al., 2002; Ricciotti and FitzGerald, 2011).”

• cites Monneret et al., 2002Not checked yet

  Not checked in the free run.

• cites Ricciotti and FitzGerald, 2011Not checked yet

  Not checked in the free run.

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

15d-PGJ2 is formed by dehydration of PGD2.

“15d-PGJ2 is the dehydration product of PGD2 (Forman et al., 1995; Kliewer et al., 1995)”

• cites Forman et al., 1995Not checked yet

  Not checked in the free run.

• cites Kliewer et al., 1995Not checked yet

  Not checked in the free run.

May combine two claims.

Dihomo-15d-PGJ2 synthesis and RAS activation are common features of senescent cells.

“we propose that the synthesis of dihomo-15d-PGJ2 and RAS activation are common features of senescent cells.”

Classified as probably not a substantial claim.

Extracellular PGD2 signals through the DP1 and DP2 receptors.

“Extracellular PGD2 signals through two receptors, DP1 (PTGDR) and DP2 (PTGDR2) (Hata and Breyer, 2004; Ricciotti and FitzGerald, 2011)”

• cites Hata and Breyer, 2004Not checked yet

  Not checked in the free run.

• cites Ricciotti and FitzGerald, 2011Not checked yet

  Not checked in the free run.

May combine two claims.

Prostaglandin-mediated RAS activation is a common and necessary feature of at least some forms of cellular senescence.

“Given that prostaglandin synthesis is required for senescence, the data indicate that prostaglandin-mediated RAS activation is a common and necessary feature of at least some forms of cellular senescence.”

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

The results may not apply to all lineages or all senescence inducers.

“Results may not reflect all lineages or inducers of senescence.”

Classified as probably not a substantial claim.

All quantitative assays from conditioned media were normalized to cell number.

“All quantitative assays (e.g. ELISAs) from CM were normalized to cell number.”

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

Except for dihomo-15d-PGJ2, most factors in Figure 1A and 1B are assigned by m/z and are only likely assignees.

“Outside of dihomo-15d-PGJ2, most of the factors in Figure 1A and 1B are assigned by m/z, and therefore are only likely assignees.”

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

All culture datasets are means of at least three experiments, and mouse datasets include at least six mice.

“All culture datasets represent means of at least 3 experiments, and mouse datasets reflect a minimum of 6 mice.”

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

Expression of specific lipid synthases is temporally regulated in pulmonary fibrosis.

“Indeed, we recently showed that expression of specific lipid synthases is temporally regulated in the context of pulmonary fibrosis, suggesting a transition from acute to more chronic stress phenotypes (Wiley et al., 2019a).”

• cites Wiley et al., 2019aNot checked yet

  Not checked in the free run.

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

The study did not apply any method to check whether its data satisfied the assumptions of the statistical tests.

“No methods were used to determine whether the data met the assumptions of the statistical approaches.”

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

The lipids described here probably undergo dynamic regulation as well.

“The lipids described here likely undergo dynamic regulation as well.”

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

All cells used were free of mycoplasma.

“All cells were mycoplasma free.”

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

The compound parameters for the HPLC-QTRAP-MS analysis were established using appropriate standards.

“The compound parameters were established using appropriate standards.”

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

The study analyzed only mice and human cells, using three cell types as representatives of endothelial, epithelial, and mesenchymal lineages.

“This study only features analyses from mice and human cells, and for 3 cell types as representatives of endothelial, epithelial, and mesenchymal lineages.”

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

Peak areas from cellular extracts were normalized to total protein or cell number.

“Peak areas were normalized to total protein or cell number.”

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

Senolytic drugs are increasingly used in aging research and have entered early clinical trials.

“Senolytic drugs are increasingly being used in aging and related research and – importantly – have entered early clinical trials (Hickson et al., 2019; Justice et al., 2019).”

• cites Hickson et al., 2019 sourceNot checked yet

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• cites Justice et al., 2019 sourceNot checked yet

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

Animal experiments were performed under a protocol approved by the Buck Institute's Institutional Animal Care and Use Committee.

“Animal experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee of the Buck Institute.”

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

Senescence and the SASP are dynamic phenotypes that change over time.

“Senescence and the SASP are dynamic phenotypes that change over time (De Cecco et al., 2019; Hernandez-Segura et al., 2017).”

• cites De Cecco et al., 2019 sourceNot checked yet

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• cites Hernandez-Segura et al., 2017 sourceNot checked yet

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