Iron accumulation in senescent cells is coupled with impaired ferritinophagy and inhibition of ferroptosis

May combine two claims.

Senescent MEFs (MEF IR) have significantly elevated levels of transferrin receptor 1 (TfR1), ferroportin, and ferritin, while divalent metal transporter 1 (DMT1) does not significantly change.

“Western blot analyses revealed that senescent MEFs (MEF IR) had significantly elevated levels of transferrin receptor 1 (TfR1), the principle protein responsible for the cellular uptake of iron via transferrin (Fe3+-transferrin) (Fig. 3A).”

• Also stated in: § Altered iron homeostatic mechanisms drive senescent cells to acquire profound levels of intracellular iron · plain

  “The divalent metal transporter 1 (DMT1) protein, which is involved in transport of iron (Fe2+) from endosomes to cytoplasm, did not significantly change (Fig. 3A).”

• Also stated in: § Altered iron homeostatic mechanisms drive senescent cells to acquire profound levels of intracellular iron · plain

  “Ferroportin was also increased in senescent cells (Fig. 3A) and can function to efflux iron from the cell under certain conditions.”

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

Senescent MEFs (MEF IR) have significantly lower levels of iron regulatory protein 1 (IRP1) and iron-sulfur cluster assembly enzyme (ISCU), and markedly elevated IRP2.

“Senescent MEFs (MEFs IR) had significantly lower levels of both IRP1 and its regulator the iron-sulfur cluster assembly enzyme (ISCU) (Fig. 3C), which is required for iron-sulfur cluster biogenesis [48], [49], [50].”

• Also stated in: § Altered iron homeostatic mechanisms drive senescent cells to acquire profound levels of intracellular iron · plain

  “senescent MEFs (MEF IR) had markedly elevated IRP2 expression, consistent with having elevated TfR1 but not with elevated ferritin and ferroportin (Fig. 3A).”

May combine two claims.

Iron accumulation in irradiated MEFs is reliant on p53-mediated senescence induction and is not indirectly caused by irradiation.

“The iron accumulation was reliant on p53-mediated senescence induction and was not indirectly caused by irradiation (Fig. S1E).”

May combine two claims.

Total cellular protein is slightly elevated (~1.3-fold) in senescent MEFs but cannot account for the ~20-fold increase in intracellular iron.

“Total cellular protein was slightly elevated (~ 1.3-fold) in senescent MEFs (MEF IR) (Fig. S1G), but could not account for the magnitude of increased intracellular iron (~ 20-fold) (Fig. 1A(iii)).”

May combine two claims.

Promoting ferritin degradation with the autophagy activator rapamycin averts the iron accumulation phenotype of senescent cells, preventing the increase of TfR1, ferritin and intracellular iron.

“Promoting ferritin degradation by using the autophagy activator rapamycin averted the iron accumulation phenotype of senescent cells, preventing the increase of TfR1, ferritin and intracellular iron, but failed to re-sensitize these cells to ferroptosis.”

May combine two claims.

Adhered senescent MEFs have approximately twice the diameter of primary non-irradiated MEFs, but suspended cell diameters are closely aligned.

“the diameter of adhered senescent MEFs (at 21 days post-irradiation) was approximately twice that of primary non-irradiated MEFs (MEF PRI) (Fig. S1F). However, diameters of suspended cells measured following dissociation of adherent cells were closely aligned (Fig. S1G).”

May combine two claims.

Immortalised MEFs (MEF-LT) transduced with HRasV12 show no signs of cellular senescence and no iron accumulation.

“Immortalised primary MEFs (MEF-LT) transduced with retroviruses containing HRasV12 showed no signs of cellular senescence and accordingly no iron accumulation (Fig. 1C(ii)).”

May combine two claims.

Senescent MEFs remain viable and can be metabolically active in culture for many months.

“Senescent MEFs remain viable (Fig. S1B) and can be metabolically active in culture for many months [2].”

• cites [2] van Deursen, 2014, The role of senescent cells in ageing, Nature sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Ferritin is substantially increased in senescent human diploid fibroblasts, senescent human prostate epithelial cells, and replicative senescent MEFs.

“these human cell lines both displayed a substantial increase in intracellular ferritin (Fig. 3B(i),(ii)), consistent with concurrent iron accumulation (Fig. 2).”

• Also stated in: § Altered iron homeostatic mechanisms drive senescent cells to acquire profound levels of intracellular iron · plain

  “when senescence in MEFs was induced through continual passaging (REP), there was likewise a marked elevation in ferritin (Fig. 3B(iii)) corresponding to the iron accumulation (Fig. 1B).”

May combine two claims.

In mouse ageing hepatic tissue, enrichment of senescent cells accompanies iron accumulation and an elevation in ferritin.

“Finally, the enrichment of senescent cells in mouse ageing hepatic tissue was found to accompany iron accumulation, an elevation in ferritin and mirrored our observations using cultured senescent cells.”

May combine two claims.

MEFs that spontaneously bypassed senescence (NIH3T3, ISO3T3) or were immortalised with SV40 large T antigen have intracellular iron levels comparable to primary MEFs.

“MEFs that underwent senescence-bypass [NIH(3T3)] had intracellular iron levels comparable to that of primary MEFs (MEF PRI).”

• Also stated in: § Senescent cells accumulate large amounts of intracellular iron · plain

  “immortalised MEFs (MEF LT) at passage 7 had intracellular iron levels comparable to that of primary MEFs (MEF PRI).”

May combine two claims.

Intracellular iron accumulation in irradiated MEFs occurs in parallel with the formation of senescent cells and plateaus between days 14 and 21 post-irradiation.

“The accumulation of intracellular iron (Fig. 4B) occurred in parallel with the formation of senescent cells and reflected the percentage of senescent cells at days 7 (>50%) and 14 (> 80%) post-irradiation (Fig. 4A).”

• Also stated in: § Cellular senescence precedes iron accumulation and provides resistance to iron induced toxicity · plain

  “However, the amount of accumulated iron between days 14 and 21 plateaued.”

May combine two claims.

Senescent cells had increased TfR1 and ferritin, coupled with reduced ferroportin at the plasma membrane.

“Senescent cells paradoxically had increased TfR1 and ferritin, coupled with reduced ferroportin at the plasma membrane (Fig. 3A,Figs. S2 and S3C).”

May combine two claims.

Senescent MEFs at 14 days post-irradiation under FAC treatment further increase TfR1 (at 5 μM FAC) and ferritin.

“under the same treatment conditions, senescent MEFs (14 days post-irradiation) further increased TfR1 (5 µM FAC) and likewise ferritin (Fig. 4E(ii)).”

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

Senescent MEFs at 14 days post-irradiation show a further dose-dependent increase in iron accumulation when treated with ferric ammonium citrate (FAC), accumulating up to ~3-fold more with 40 μM FAC than senescent cells in basal medium and ~30-fold more than primary MEFs in basal medium.

“there was a further dose-dependent increase in the capacity of senescent MEFs to accumulate iron, with cells accumulating up to ~ 3-fold more (with 40 µM FAC) when compared with senescent cells cultured in basal medium and ~ 30-fold more when compared with primary MEFs cultured in basal medium (Fig. 4D).”

May combine two claims.

LC3-II also accumulated in replicative senescent MEFs (MEF REP), concomitant with elevated ferritin.

“LC3-II also accumulated in replicative senescent MEFs (MEF REP), concomitant with elevated ferritin (Fig. S3C).”

May combine two claims.

Primary MEFs with augmented intracellular iron following 24 h FAC treatment have reduced TfR1 and upregulated ferritin.

“primary MEFs with augmented intracellular iron (following 24 h FAC treatments) (Fig. 4D) had reduced TfR1 and upregulated ferritin in order to counteract toxicity (Fig. 4E(i)).”

May combine two claims.

Stored iron in senescent MEFs, once accumulated, is highly resistant to chelation, with 24 h deferiprone treatment causing only a marginal decrease in intracellular iron and a negligible effect on ferritin.

“Stored iron in senescent MEFs once accumulated is highly resistant to chelation.”

• Also stated in: § Cellular senescence precedes iron accumulation and provides resistance to iron induced toxicity · plain

  “Senescent MEFs (21 days post-irradiation) treated with Dfp (50 µM) for 24 h showed (i) only a marginal decrease in intracellular iron as determined by ICP-MS.”

• Also stated in: § Cellular senescence precedes iron accumulation and provides resistance to iron induced toxicity · boosted

  “Western blot analyses and densitometry showed a negligible effect of Dfp treatment on ferritin level of senescent MEFs.”

May combine two claims.

Senescent cells had barely detectable ISCU and markedly reduced IRP1.

“However, while senescent cells had barely detectable ISCU, consistent with deficient cellular iron, this was coupled with markedly reduced IRP1”

May combine two claims.

Ferritin levels remained responsive in senescent cells, further increasing in iron supplemented media and affording greater tolerance to iron-induced toxicity.

“Furthermore, ferritin levels remained responsive in senescent cells, further increasing in iron supplemented media and affording greater tolerance to iron-induced toxicity (Fig. 4E(ii), F).”

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

Removal of p16-positive senescent cells in ageing mice delays tumourigenesis and attenuates age-related deterioration of several organs without adverse side effects.

“Removal of p16-positive senescent cells in ageing mice delayed tumourigenesis and attenuated age-related deterioration of several organs, including kidney, heart and adipose tissue, without adverse side effects [3].”

• cites [3] Baker, 2016, Naturally occurringp16(Ink4a)-positive cells shorten healthy lifespan, Nature sourceNot checked yet

  Not checked in the free run.

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

Removal of p16-positive senescent cells in mice extends median lifespan.

“Furthermore, a median lifespan extension was observed, indicating that p16-positive senescent cells negatively impact longevity [3].”

• cites [3] Baker, 2016, Naturally occurringp16(Ink4a)-positive cells shorten healthy lifespan, Nature sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Ferritin may be a marker of cellular senescence and associated iron accumulation.

“Therefore, ferritin may be a marker of cellular senescence and associated iron accumulation.”

May combine two claims.

Impaired ferritin degradation explains the iron accumulation phenotype of senescent cells, whereby iron is effectively trapped in ferritin creating a perceived cellular deficiency.

“Impaired ferritin degradation explains the iron accumulation phenotype of senescent cells, whereby iron is effectively trapped in ferritin creating a perceived cellular deficiency.”

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

The iron storage protein ferritin is enriched in ageing tissues.

“Iron accumulation occurs in replicative senescent fibroblasts in vitro [35] and the iron storage protein ferritin is enriched in ageing tissues [36], [37].”

• cites [36] Ott, 2016, Macroautophagy is impaired in old murine brain tissue as well as in senescent human fibroblasts, Redox Biol. sourceNot checked yet

  Not checked in the free run.

• cites [37] DeRuisseau, 2013, Aging-related changes in the iron status of skeletal muscle, Exp. Gerontol. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Iron accumulation in senescent cells is driven by impaired ferritinophagy, a lysosomal process that promotes ferritin degradation and ferroptosis.

“Iron accumulation in senescent cells was driven by impaired ferritinophagy, a lysosomal process that promotes ferritin degradation and ferroptosis.”

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

Age-dependent accumulation of iron in various tissues occurs separately to the enrichment of senescent cells.

“Age-dependent accumulation of iron in various tissues has been reported to occur separately to the enrichment of senescent cells [11], [12], [16], [17], [18], [19].”

• cites [11] Fairweather-Tait, 2014, Iron status in the elderly, Mech. Ageing Dev. sourceNot checked yet

  Not checked in the free run.

• cites [12] Xu, 2012, Impaired iron status in aging research, Int. J. Mol. Sci. sourceNot checked yet

  Not checked in the free run.

• cites [16] Hahn, 2009, Age-dependent and gender-specific changes in mouse tissue iron by strain, Exp. Gerontol. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Diseases associated with the accumulation of senescent cells, such as neurodegenerative disorders, osteoarthritis and idiopathic pulmonary fibrosis, exhibit iron dyshomeostasis, where iron burden often correlates with disease severity.

“Diseases associated with the accumulation of senescent cells, such as neurodegenerative disorders (e.g. Alzheimer's and Parkinson's) [20], [21], [22], osteoarthritis [23], [24] and idiopathic pulmonary fibrosis [25], also exhibit iron dyshomeostasis where often iron burden correlates with disease severity [14], [15], [26], [27], [28], [29], [30], [31], [32], [33], [34].”

• cites [20] Chinta, 2015, Cellular senescence and the aging brain, Exp. Gerontol. sourceNot checked yet

  Not checked in the free run.

• cites [21] Bhat, 2012, Astrocyte senescence as a component of Alzheimer's disease, PLoS One sourceNot checked yet

  Not checked in the free run.

• cites [22] Chinta, 2013, Environmental stress, ageing and glial cell senescence: a novel mechanistic link to Parkinson's disease?, J. Intern. Med. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Ferritin levels serve as a robust biomarker of cellular senescence, associated iron accumulation, and resistance to iron-induced toxicity.

“For instance, ferritin (iron storage) levels provided a robust biomarker of cellular senescence, for associated iron accumulation and for resistance to iron-induced toxicity.”

• Also stated in: § Altered iron homeostatic mechanisms drive senescent cells to acquire profound levels of intracellular iron · hedged

  “Therefore, ferritin may be a marker of cellular senescence and associated iron accumulation.”

May combine two claims.

Homeostatic mechanisms in senescent MEFs are geared for continual uptake and sequestration of milieu iron and afford resistance to toxicity from excess iron.

“Therefore, homeostatic mechanisms in senescent MEFs are geared for continual uptake and sequestration of milieu iron and afford cells resistance to toxicity from excess iron.”

• AssumptionRests on an assumption: Total intracellular iron measured by ICP-MS reflects the iron pool relevant to ferroptosis and iron-induced toxicity.

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

Senescent cells can impair tissue function in ageing despite their relatively low abundance (<15%).

“Senescent cells can impair tissue function in ageing despite their relatively low abundance (< 15%) [3], [4].”

• cites [3] Baker, 2016, Naturally occurringp16(Ink4a)-positive cells shorten healthy lifespan, Nature sourceNot checked yet

  Not checked in the free run.

• cites [4] Baker, 2011, Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders, Nature sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Ferroportin is unlikely to participate in effective iron efflux, and iron accumulation prevails through enhanced uptake and sequestration in senescent cells.

“Ferroportin is therefore unlikely to participate in effective iron efflux and consequently iron accumulation prevails through enhanced uptake and sequestration.”

May combine two claims.

Lysosomal impairment is a feature of cellular senescence and is associated with ferritin accumulation (impaired ferritinophagy) and resistance to ferroptosis.

“Taken together, these results demonstrate that lysosomal impairment is a feature of cellular senescence and is associated with ferritin accumulation (impaired ferritinophagy) and resistance to ferroptosis.”

May combine two claims.

Ferritinophagy is important for liberating iron from ferritin and for maintaining cellular iron homeostasis.

“Ferritinophagy, the process of ferritin being sequestered into autophagosomes and delivered to lysosomes for degradation, is important for liberating iron from ferritin and thus for maintaining cellular iron homeostasis [51], [53], [54], [55].”

• cites [51] Asano, 2011, Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells, Mol. Cell. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [53] Mancias, 2014, Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy, Nature sourceNot checked yet

  Not checked in the free run.

• cites [54] Kidane, 2006, Release of iron from ferritin requires lysosomal activity, Am. J. Physiol. Cell Physiol. sourceNot checked yet

  Not checked in the free run.

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

In mammalian cells, ferritins assemble to form large (480 kDa) complexes with cavities capable of accommodating up to 4500 atoms of iron and function to modulate iron bioavailability and detoxification.

“In mammalian cells, ferritins assemble to form large (480 kDa) complexes with cavities capable of accommodating up to 4500 atoms of iron and function to modulate iron bioavailability and detoxification within cells [47].”

• cites [47] MacKenzie, 2008, Intracellular iron transport and storage: from molecular mechanisms to health implications, Antioxid. Redox Signal. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Cellular senescence is associated with impaired ferritinophagy, explaining the associated iron-accumulation phenotype.

“Together these results demonstrate that cellular senescence is associated with impaired ferritinophagy, explaining the associated iron-accumulation phenotype.”

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

Increased cellular iron uptake positively regulates ferritin expression at transcriptional and translational levels to protect from labile iron-mediated oxidative stress.

“Increased cellular iron uptake positively regulates ferritin expression, both at the transcriptional and translational levels, to afford protection from labile iron-mediated oxidative stress [38].”

• cites [38] Freund, 2012, Lamin B1 loss is a senescence-associated biomarker, Mol. Biol. Cell. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Ferritin may provide a robust biomarker for cellular senescence and for associated iron accumulation.

“which may provide a robust biomarker for cellular senescence and for associated iron accumulation (Figs. 3A,B,4C,E,6C,D).”

May combine two claims.

The enrichment of senescent cells in ageing hepatic tissue was found to accompany iron accumulation, an elevation in ferritin, and mirrored in vitro observations using cultured senescent cells.

“Taken together, the enrichment of senescent cells in ageing hepatic tissue was found to accompany iron accumulation, an elevation in the iron-storage ferritin protein and mirrored our observations in vitro using cultured senescent cells.”

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

Most cells subsequently down-regulate iron uptake mechanisms, particularly TfR1, and up-regulate iron efflux via ferroportin to regain homeostatic levels.

“Most cells subsequently down-regulate iron uptake mechanisms, particularly TfR1 and up-regulate iron efflux via ferroportin in order to regain homeostatic levels (Fig. 4E(i)) [47].”

• cites [47] MacKenzie, 2008, Intracellular iron transport and storage: from molecular mechanisms to health implications, Antioxid. Redox Signal. sourceNot checked yet

  Not checked in the free run.

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

Rapamycin selectively activates autophagy through inhibiting the mTOR pathway and can extend the lifespan of mice.

“Rapamycin selectively activates autophagy through inhibiting the mTOR pathway [60] and can extend the lifespan of mice [79].”

• cites [60] Sarkar, 2009, Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies, Cell Death Differ. sourceNot checked yet

  Not checked in the free run.

• cites [79] Harrison, 2009, Rapamycin fed late in life extends lifespan in genetically heterogeneous mice, Nature sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Senescent cells having perturbed ferritin degradation sequester iron (trapped in ferritin), creating a perpetual state of deficiency and a cycle promoting continual iron acquisition.

“However, senescent cells having perturbed ferritin degradation sequester iron (trapped in ferritin), creating a perpetual state of deficiency and a cycle promoting continual iron acquisition.”

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

Erastin induces ferroptosis through silencing SLC7A11 and enhances iron bioavailability through autophagic/lysosomal degradation of ferritin and NCOA4.

“Erastin induces ferroptosis through silencing SLC7A11 [84] and was recently demonstrated to enhance iron bioavailability through autophagic/lysosomal degradation of ferritin and its cargo receptor NCOA4 [63].”

• cites [84] Dixon, 2014, Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis, eLife sourceNot checked yet

  Not checked in the free run.

• cites [63] Gao, 2016, Ferroptosis is an autophagic cell death process, Cell Res. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Ferritin is selectively targeted for autophagic/lysosomal degradation by the cargo receptor NCOA4, a process required for liberating iron making it bioavailable.

“Ferritin is selectively targeted for autophagic/lysosomal degradation by the cargo receptor NCOA4 [53], a process required for liberating iron making it bioavailable to the cell [51], [53], [65].”

• cites [53] Mancias, 2014, Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy, Nature sourceNot checked yet

  Not checked in the free run.

• cites [51] Asano, 2011, Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells, Mol. Cell. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [65] Hou, 2016, Autophagy promotes ferroptosis by degradation of ferritin, Autophagy sourceNot checked yet

  Not checked in the free run.

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

RSL3 induces ferroptosis through direct inhibition of GPX4 without dependence upon system Xc- inhibition or glutathione depletion.

“which in contrast to erastin induces ferroptosis through direct inhibition of glutathione peroxidase 4 (GPX4) without dependance upon system Xc- inhibition or glutathione depletion [82].”

• cites [82] Cao, 2016, Mechanisms of ferroptosis, Cell. Mol. Life Sci.: CMLS sourceNot checked yet

  Not checked in the free run.

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

Rapamycin treatment may lead to autophagic degradation of TfR1 or modulate levels by enhancing degradation of TfR1 mRNA.

“Additionally, rapamycin treatment may lead to autophagic degradation of TfR1 or modulate levels by enhancing degradation of TfR1 mRNA [78].”

• cites [78] Bayeva, 2012, mTOR regulates cellular iron homeostasis through tristetraprolin, Cell Metab. sourceNot checked yet

  Not checked in the free run.

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

Autophagy involves delivery of cytoplasmic substrates to lysosomes for degradation, and impairment of this process is associated with premature ageing and age-associated decline in tissue function.

“Autophagy involves the delivery of cytoplasmic substrates (cellular proteins and damaged organelles) to lysosomes for degradation and impairment of this process is associated with premature ageing and age-associated decline in tissue function [58], [71].”

• cites [58] Tanida, 2005, Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy, Autophagy sourceNot checked yet

  Not checked in the free run.

• cites [71] Rubinsztein, 2011, Autophagy and aging, Cell sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Impaired ferritinophagy traps iron in ferritin, creating a perceived cellular deficiency, which causes concomitant changes in levels of iron regulatory proteins.

“Impaired ferritinophagy traps iron in ferritin creating a perceived cellular deficiency, which in turn causes concomitant changes in the levels of iron regulatory proteins.”

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

Ferritin and ferroportin can be regulated through mRNA stability or post-translationally through protein turnover.

“Ferritin and ferroportin can be regulated through mRNA stability or post-translationally through protein turnover [85], [86].”

• cites [85] Ward, 2012, Ferroportin-mediated iron transport: expression and regulation, Biochim. Biophys. Acta sourceNot checked yet

  Not checked in the free run.

• cites [86] Drysdale, 1966, Regulation of synthesis and turnover of ferritin in rat liver, J. Biol. Chem. sourceNot checked yet

  Not checked in the free run.

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

Impaired autophagy in senescent cells has previously been shown to reduce ferritin turnover, but intracellular iron levels or major iron homeostasis proteins were not evaluated in that study.

“Coincidently, impaired autophagy in senescent cells has previously been shown to reduce ferritin turnover, but in this study intracellular iron levels or the levels of major iron homeostasis proteins were not evaluated [77].”

• cites [77] Ott, 2016, Reduced autophagy leads to an impaired ferritin turnover in senescent fibroblasts, Free Radic. Biol. Med. sourceNot checked yet

  Not checked in the free run.

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

Lysosomal dysfunction can precede senescence development and drive associated phenotypic changes.

“Additionally, lysosomal dysfunction can precede senescence development and drive the associated phenotypic changes [57], [73].”

• cites [57] Tai, 2017, Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence, Autophagy sourceNot checked yet

  Not checked in the free run.

• cites [73] Kang, 2011, Autophagy impairment induces premature senescence in primary human fibroblasts, PLoS One sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Lysosomal dysfunction in senescent cells can account for ferritin and ferroportin accumulation, as both proteins are degraded through the autophagic pathway.

“Nonetheless, lysosomal dysfunction in senescent cells can account for ferritin and ferroportin accumulation, as both proteins are degraded through the autophagic pathway [51], [89].”

• cites [51] Asano, 2011, Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells, Mol. Cell. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [89] Nemeth, 2004, Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization, Science sourceNot checked yet

  Not checked in the free run.

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

Ferroptosis can be initiated through repression of SLC7A11, resulting in iron-dependent accumulation of ROS and glutathione depletion.

“Distinctly, ferroptosis can be initiated through repression of the p53 target, SLC7A11, which encodes a component of the cysteine/glutamate antiporter (system Xc-), resulting in iron-dependent accumulation of ROS and glutathione (antioxidant) depletion [62], [82].”

• cites [62] Dixon, 2012, Ferroptosis: an iron-dependent form of nonapoptotic cell death, Cell sourceNot checked yet

  Not checked in the free run.

• cites [82] Cao, 2016, Mechanisms of ferroptosis, Cell. Mol. Life Sci.: CMLS sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Depletion of LAMP2 can induce cellular senescence, as can pharmacological inhibition of lysosomal activity.

“Depletion of lysosomal-associated membrane protein 2 (LAMP2), which is critical for fusion between autophagosomes and lysosomes, can induce cellular senescence [57], [73], as can pharmacological inhibition of lysosomal activity (hydroxychloroquine & leupeptin treatment) [57].”

• cites [57] Tai, 2017, Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence, Autophagy sourceNot checked yet

  Not checked in the free run.

• cites [73] Kang, 2011, Autophagy impairment induces premature senescence in primary human fibroblasts, PLoS One sourceNot checked yet

  Not checked in the free run.

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

IRP1 usually loses its iron-sulfur cluster and aconitase activity and binds IREs with high affinity.

“In this situation, IRP1 usually loses its iron-sulfur cluster and aconitase activity and binds IREs with high affinity [48], [50].”

• cites [48] Rouault, 2006, The role of iron regulatory proteins in mammalian iron homeostasis and disease, Nat. Chem. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [50] Rouault, 2015, Mammalian iron-sulphur proteins: novel insights into biogenesis and function, Nat. Rev. Mol. Cell Biol. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Lysosomal dysfunction has been documented in replicative and oxidative stress-induced senescence and in both cases was associated with buildup of autophagosomes and related markers (e.g., LC3-II, p62).

“Consistent with our findings (Fig. 5,Fig. S3), lysosomal dysfunction has been documented in replicative [74] and oxidative stress-induced senescence [57] and in both cases was associated with the buildup of autophagosomes and related markers (e.g. LC3-II, p62).”

• cites [74] Lin, 2015, Downregulation of dynamin-related protein 1 contributes to impaired autophagic flux and angiogenic function in senescent endothelial cells, Arterioscler. Thromb. Vasc. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [57] Tai, 2017, Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence, Autophagy sourceNot checked yet

  Not checked in the free run.

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

GATA4 activates NF-kB to initiate SASP and facilitate senescence progression.

“GATA4 in turn activates the NF-kB transcription factor to initiate SASP and facilitate senescence progression.”

• cites [76] Kang, 2015, The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4, Science sourceNot checked yet

  Not checked in the free run.

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

Autophagy impairment incurred through lysosomal dysfunction has been described in cellular senescence and shown to regulate SASP.

“Autophagy impairment incurred through lysosomal dysfunction has recently been described in cellular senescence and shown to regulate SASP [57], [72], [73], [74], [75].”

• cites [57] Tai, 2017, Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence, Autophagy sourceNot checked yet

  Not checked in the free run.

• cites [72] Han, 2016, AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation, Aging Cell sourceNot checked yet

  Not checked in the free run.

• cites [73] Kang, 2011, Autophagy impairment induces premature senescence in primary human fibroblasts, PLoS One sourceNot checked yet

  Not checked in the free run.

May combine two claims.

IRP2 can mediate degradation of ferritin and ferroportin mRNA, but this must be insufficient to prevent their biosynthesis in senescent cells.

“While IRP2 can mediate the degradation of ferritin and ferroportin mRNA [48], this must be insufficient to prevent their biosynthesis in the context of senescent cells.”

• cites [48] Rouault, 2006, The role of iron regulatory proteins in mammalian iron homeostasis and disease, Nat. Chem. Biol. sourceNot checked yet

  Not checked in the free run.

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

Cell death elicited by ferroptosis-inducing compounds (e.g., erastin and RSL3) can be mitigated through various iron chelators.

“Cell death elicited by ferroptosis-inducing compounds (FINs) (e.g. erastin and RSL3) can also be mitigated through various iron chelators (Dfo, 2,2-bipyridile or compound 311) [62], [66], [81].”

• cites [62] Dixon, 2012, Ferroptosis: an iron-dependent form of nonapoptotic cell death, Cell sourceNot checked yet

  Not checked in the free run.

• cites [66] Yang, 2008, Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells, Chem. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [81] Reed, 2012, Ironing out cell death mechanisms, Cell sourceNot checked yet

  Not checked in the free run.

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

ISCU levels are suppressed by iron deprivation, to decrease iron utilization by curtailing iron-sulfur cluster biogenesis.

“Additionally, ISCU levels are suppressed by iron deprivation, to decrease iron utilization by curtailing iron-sulfur cluster biogenesis [90].”

• cites [90] Tong, 2006, Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis, Cell Metab. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

This scenario accounts for the profound iron accumulation observed in senescent cells (up to 20-fold) and for their capacity to extract further iron from enriched media.

“This scenario accounts for the profound iron accumulation observed in senescent cells (up to 20-fold) and for their capacity to extract further iron from enriched media (Fig. 4E(ii)).”

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

Cells are desensitized to ferroptosis through genetic silencing of iron uptake (TfR1), metabolism (IRP2 and ISCU) and storage (ferritin) genes, or through depletion of transferrin in the extracellular environment.

“Cells are also desensitized to ferroptosis through genetic silencing of iron uptake (TfR1), metabolism (IRP2 and ISCU) and storage (ferritin) genes [62], [66], [82], or through depletion of transferrin in the extracellular environment [83].”

• cites [62] Dixon, 2012, Ferroptosis: an iron-dependent form of nonapoptotic cell death, Cell sourceNot checked yet

  Not checked in the free run.

• cites [66] Yang, 2008, Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells, Chem. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [82] Cao, 2016, Mechanisms of ferroptosis, Cell. Mol. Life Sci.: CMLS sourceNot checked yet

  Not checked in the free run.

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

Ferroptosis is a unique cell death pathway distinct from apoptosis, reliant on availability of iron liberated through ferritinophagy.

“Ferroptosis, a unique cell death pathway distinct from apoptosis [62], [66], [80], is reliant on the availability of iron [62], [66], [81] liberated through ferritinophagy [63].”

• cites [62] Dixon, 2012, Ferroptosis: an iron-dependent form of nonapoptotic cell death, Cell sourceNot checked yet

  Not checked in the free run.

• cites [66] Yang, 2008, Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells, Chem. Biol. sourceNot checked yet

  Not checked in the free run.

• cites [80] Yagoda, 2007, RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels, Nature sourceNot checked yet

  Not checked in the free run.

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

The mTOR pathway is a negative regulator of autophagy and is implicated in cellular senescence, notably through SA-βgal activity and expression of p16 and p21 markers.

“The mTOR pathway is a negative regulator of autophagy and is implicated in cellular senescence [67], most notably through SA-βgal activity and the expression of p16 and p21 markers [67], [68].”

• cites [67] Xu, 2014, mTOR signaling from cellular senescence to organismal aging, Aging Dis.Not checked yet

  Not checked in the free run.

• cites [68] Laberge, 2015, MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation, Nat. Cell Biol. sourceNot checked yet

  Not checked in the free run.

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

Bafilomycin A1 is an inhibitor of late-phase autophagy that prevents fusion of autophagosomes and lysosomes.

“bafilomycin A1 (100 nM), an inhibitor of late-phase autophagy (prevents fusion of autophagosomes and lysosomes) [61]”

• cites [61] Yamamoto, 1998, Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells, Cell Struct. Funct. sourceNot checked yet

  Not checked in the free run.

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

The ferritin antibody was diluted in these experiments to facilitate the exclusive detection of ferritin overexpressing cells.

“Note that the ferritin antibody was diluted in these experiments to facilitate the exclusive detection of ferritin overexpressing cells.”

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

Quadruplicate determinations of iron concentration were performed for each sample.

“Quadruplicate determinations of iron concentration was performed for each sample.”

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

Control non-irradiated cell lines were cultured to equivalent confluence and otherwise handled in the same manner as irradiated cells.

“Control non-irradiated cell lines were cultured to equivalent confluence and otherwise handled in the same manner as irradiated cells.”

Classified as probably not a substantial claim.

Loss of pS6 promotes autophagosome formation.

“Loss of pS6 promotes autophagosome formation [69].”

• cites [69] Blommaart, 1995, Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes, J. Biol. Chem. sourceNot checked yet

  Not checked in the free run.

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

At least three independent experiments were used for all comparisons.

“At least three independent experiments were used for all comparisons.”

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

This study was approved by the Deakin University Animal Ethics Committee (AEC) (Id#G01-2014).

“This study was approved by the Deakin University Animal Ethics Committee (AEC) (Id#G01-2014).”

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

Rapamycin (100 nM) selectively activates autophagy through inhibiting the mTOR pathway.

“We used rapamycin (100 nM), which selectively activates autophagy through inhibiting the mTOR pathway [60] to activate autophagy in primary MEFs (MEF PRI), significantly increasing the LC3-II: LC3-I ratio (Fig. 5C).”

• cites [60] Sarkar, 2009, Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies, Cell Death Differ. sourceNot checked yet

  Not checked in the free run.

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

Erastin and RSL3 are two structurally different inducers of ferroptosis that affect distinct pathways.

“two structurally different inducers of ferroptosis that affect distinct pathways [66].”

• cites [66] Yang, 2008, Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells, Chem. Biol. sourceNot checked yet

  Not checked in the free run.

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

Elevated p62 levels were previously observed in oxidative stress-induced senescent cells.

“Elevated p62 levels were previously observed in oxidative stress-induced senescent cells [57], and consistent with this study, we demonstrated that our senescent MEFs (MEF IR) likewise had markedly elevated levels of p62 when compared to primary MEFs (MEF PRI) (Fig. 5D).”

• cites [57] Tai, 2017, Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence, Autophagy sourceNot checked yet

  Not checked in the free run.

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

Ferroptosis is an iron-dependent autophagic cell death program that can be inhibited in MEFs by genetically inactivating autophagy or ferritinophagy.

“an iron-dependent autophagic cell death program [62], [63], [64] that can be inhibited in MEFs by genetically inactivating autophagy or ferritinophagy [65].”

• cites [62] Dixon, 2012, Ferroptosis: an iron-dependent form of nonapoptotic cell death, Cell sourceNot checked yet

  Not checked in the free run.

• cites [63] Gao, 2016, Ferroptosis is an autophagic cell death process, Cell Res. sourceNot checked yet

  Not checked in the free run.

• cites [64] Wolpaw, 2011, Modulatory profiling identifies mechanisms of small molecule-induced cell death, Proc. Natl. Acad. Sci. USA sourceNot checked yet

  Not checked in the free run.