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. 2018 Apr:14:100-115.
doi: 10.1016/j.redox.2017.08.015. Epub 2017 Sep 1.

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

Shashank Masaldan  1 , Sharnel A S Clatworthy  1 , Cristina Gamell  2 , Peter M Meggyesy  1 , Antonia-Tonia Rigopoulos  1 , Sue Haupt  3 , Ygal Haupt  4 , Delphine Denoyer  1 , Paul A Adlard  5 , Ashley I Bush  5 , Michael A Cater  6
Affiliations

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

Shashank Masaldan et al. Redox Biol. 2018 Apr.

Abstract

Cellular senescence is characterised by the irreversible arrest of proliferation, a pro-inflammatory secretory phenotype and evasion of programmed cell death mechanisms. We report that senescence alters cellular iron acquisition and storage and also impedes iron-mediated cell death pathways. Senescent cells, regardless of stimuli (irradiation, replicative or oncogenic), accumulate vast amounts of intracellular iron (up to 30-fold) with concomitant changes in the levels of iron homeostasis proteins. For instance, ferritin (iron storage) levels provided a robust biomarker of cellular senescence, for associated iron accumulation and for resistance to iron-induced toxicity. Cellular senescence preceded iron accumulation and was not perturbed by sustained iron chelation (deferiprone). Iron accumulation in senescent cells was driven by impaired ferritinophagy, a lysosomal process that promotes ferritin degradation and ferroptosis. Lysosomal dysfunction in senescent cells was confirmed through several markers, including the build-up of microtubule-associated protein light chain 3 (LC3-II) in autophagosomes. Impaired ferritin degradation explains the iron accumulation phenotype of senescent cells, whereby iron is effectively trapped in ferritin creating a perceived cellular deficiency. Accordingly, senescent cells were highly resistant to ferroptosis. 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. 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.

Keywords: Ageing; Autophagy; Ferritin; Ferritinophagy; Ferroptosis; Iron; Senescence.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Senescent MEFs accumulate vast amounts of intracellular iron. (A) Induction of senescence in MEFs by sublethal gamma irradiation (10 Gy) caused intracellular iron accumulation. (i) Percentage of senescent MEFs in primary (MEF PRI) and irradiated (MEF IR) cultures as determined by SA-βgal activity. The majority (> 80%) of MEFs displayed positive SA-βgal activity 10 days post-irradiation (IR) (blue staining). Images were taken at 100× magnification. (ii) Western blot analyses of molecular markers of senescence, p53 and p16, in primary (PRI) and irradiated (IR) MEFs. Senescent MEFs (MEF IR) had elevated levels of both p53 and p16 at 21 days post-irradiation (10 Gy). β-actin was detected as a loading control. (iii) ICP-MS analyses demonstrated that senescent MEFs (MEF IR) accumulated intracellular iron (~ 20-fold) at 21 days post-irradiation (10 Gy). Note that media were replenished weekly. (B) Induction of senescence in MEFs by replicative exhaustion caused intracellular iron accumulation. (i) Percentage of senescent MEFs in primary (MEF PRI), replicative senescent (MEF REP) and senescence-bypassed NIH(3T3) cultures. The majority (> 80%) of MEFs became replicative senescent (REP) at passage 7 (10-days post seeding), being positive for SA-βgal activity (blue staining). Senescence-bypassed NIH(3T3) cells had no detectable SA-βgal activity. Images were taken at 100× magnification. (ii) Western blot analyses of molecular markers of senescence, p53 and p16, in primary (MEF PRI), replicative senescent (MEF REP) and senescence-bypassed [NIH(3T3)] MEFs. Replicative senescent MEFs (MEF REP) when cultured for 21 days at passage 7 had elevated levels of both p53 and p16. β-actin was detected as a loading control. (iii) ICP-MS analyses demonstrated that replicative senescent MEFs (REP) when cultured for 21 days at passage 7 accumulated intracellular iron (~ 20-fold). MEFs that underwent senescence-bypass [NIH(3T3)] had intracellular iron levels comparable to that of primary MEFs (MEF PRI). (iv) ICP-MS analyses demonstrated that isogenic MEFs that we aided to spontaneously bypass senescent [ISO(3T3)] also had intracellular iron level comparable to that of primary MEFs (MEF PRI). (v) Primary MEFs were immortalised with virus containing the SV40 large T antigen (LT). Western blot analyses confirmed LT expression at passage 7 post-immortalisation. β-actin was detected as a loading control. (vi) ICP-MS analyses demonstrated that immortalised MEFs (MEF LT) at passage 7 had intracellular iron levels comparable to that of primary MEFs (MEF PRI). (C) Induction of senescence in MEFs with virus containing the oncogene H-RasV12 caused intracellular iron accumulation. (i) Percentage of senescent MEFs in primary (PRI) and oncogenic-induced senescent MEFs (OIS) as determined by SA-βgal activity. MEFs transduced with virus containing H-RasV12 (OIS) were enriched for SA-βgal positive cells (> 50%) at 8 days post-transduction in comparison to MEFs transduced with control virus (VC) ( < 5%). Images were taken at 100× magnification. (ii) ICP-MS analyses demonstrated that oncogenic-induced senescent MEFs (OIS) accumulated intracellular iron (~ 4.5-fold) at 8 days post-transduction with virus containing H-RasV12. Immortalised MEFs [LT(SV40)] transduced with virus containing H-RasV12 (MEF LT Ras) had intracellular iron levels comparable to that of primary MEFs (PRI). Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
Fig. 2
Fig. 2
Human senescent cells from different linages (fibroblast or epithelial) accumulate vast amounts of intracellular iron. (A) Induction of senescence in human diploid fibroblasts and human prostate epithelial cells by irradiation (IR, 10 Gy) caused intracellular iron accumulation. (i) Percentage of senescent diploid fibroblasts in primary (HDF PRI) and irradiated (HDF IR) cultures as determined by SA-βgal activity. The majority (> 80%) of diploid fibroblasts displayed positive SA-βgal activity 10 days post-irradiation (blue staining). Images were taken at 100× magnification. (ii) ICP-MS analyses demonstrated that senescent diploid fibroblasts (HDF IR) accumulated intracellular iron (~ 3.6-fold) at 21 days post-irradiation. (iii) Percentage of senescent human prostate epithelial cells in primary (PrEC PRI) and irradiated (PrEC IR) cultures as determined by SA-βgal activity. The majority (> 80%) of prostate epithelial cells displayed positive SA-βgal activity 10 days post-irradiation (blue staining). Images were taken at 100× magnification. (iv) ICP-MS analyses demonstrated that senescent prostate epithelial cells (PrEC IR) accumulated intracellular iron (~ 8.4-fold) at 21 days post-irradiation. (B) Induction of senescence in human diploid fibroblasts and human prostate epithelial cells by replicative exhaustion caused intracellular iron accumulation. (i) Percentage of senescent diploid fibroblasts in primary (HDF PRI) and replicatively exhausted (HDF REP) cultures as determined by SA-βgal activity. The majority (> 80%) of diploid fibroblasts became replicative senescent (HDF REP) at passage 29, being positive for SA-βgal activity (blue staining). Images were taken at 100× magnification. (ii) ICP-MS analyses demonstrated that senescent diploid fibroblasts (HDF REP) when cultured for 21 days at passage 29 accumulated intracellular iron (~ 3.3-fold). (iii) Percentage of senescent prostate epithelial cells in primary (PrEC PRI) and replicatively exhausted (PrEC REP) cultures as determined by SA-βgal activity. The majority (> 80%) of prostate epithelial cells became replicative senescent (PrEC REP) at passage 6, being positive for SA-βgal activity (blue staining). Images were taken at 100× magnification. (ii) ICP-MS analyses demonstrated that senescent prostate epithelial cells (PrEC REP) when cultured for 21 days at passage 6 accumulated intracellular iron (~7.3-fold). Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
Fig. 3
Fig. 3
Altered iron homeostatic mechanisms drive senescent cells to acquire intracellular iron. (A) Expression of key iron homeostasis proteins was analysed in senescent MEFs (IR) at 21 days post-irradiation (10 Gy) by western blot and densitometry. The levels of transferrin receptor 1 (TfR1) [main iron (Fe3+) importer], divalent metal transporter 1 (DMT1) (cytosolic iron importer), ferroportin (iron exporter) and ferritin (intracellular iron storage) were measured in comparison to primary (PRI) MEFs. β-actin was detected as a loading control. (B) Western blot analyses and densitometry confirm elevated expression of ferritin in (i) senescent human diploid fibroblasts (HDF IR) and in (ii) senescent human prostate epithelial cells (PrEC IR), both at 21 days post-irradiation (10 Gy). Furthermore, elevated ferritin expression was confirmed in (iii) replicative senescent MEFs (P7) cultured for 21 days. β-actin was detected as loading controls. (C) Expression of key regulatory proteins of iron homeostasis was analysed in senescent MEFs (IR) at day 21 post-irradiation (10 Gy) by western blot and densitometry. The levels of iron regulatory protein 1 (IRP1) and iron-sulfur cluster assembly enzyme (ISCU) were found significantly lower than those in primary (PRI) MEFs, while IRP2 was significantly elevated. β-actin was detected as a loading control. Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
Fig. 4
Fig. 4
Cellular senescence precedes iron accumulation and provides resistance to iron induced toxicity. (A) Percentage of senescent MEFs (MEF IR) post-irradiation (10 Gy) over the culturing time points indicated (day 1, 7, 14 and 21), as determined by SA-βgal activity (blue staining). Images were taken at 100× magnification. (B) ICP-MS analyses demonstrated cumulative intracellular iron accumulation in irradiated (10 Gy) senescent MEFs (MEF IR) over the culturing time points indicated (day 1, 7, 14 and 21). Note that iron accumulation somewhat plateaued after day 14, but was maintained. Baseline intracellular iron level in primary MEFs (MEF PRI) is also shown. (C) Expression of iron uptake and storage proteins was analysed in senescent MEFs (IR) post-irradiation (10 Gy) over the culturing time points indicated (day 1, 7, 14 and 21) by western blot. The levels of transferrin receptor 1 (TfR1) [main iron (Fe3+) importer] and ferritin (intracellular iron storage) were determined in comparison to primary (PRI) MEFs. β-actin was detected as a loading control. (D) Capacity of senescent MEFs to accumulate iron is dependent on milieu iron concentration. Primary (PRI) MEFs or irradiated (10 Gy) senescent MEFs (IR) (cultured for 14 days) were cultured for 24 h in basal medium, or media supplemented with either 5 µM or 40 µM iron (FAC). ICP-MS analyses revealed a dose-dependent increase in accumulated iron in primary and senescent MEFs (IR) following iron treatment. (E) Senescent MEFs are geared to sequester milieu iron. (i) Western blot analyses demonstrated that TfR1 was downregulated and ferritin upregulated (fold change indicated by densitometry) when primary MEFs (MEF PRI) were cultured for 24 h in media supplemented with either 5 µM or 40 µM iron (FAC). (ii) Western blot analyses and densitometry demonstrated that TfR1 and ferritin are further upregulated when senescent cells (14 days post-irradiation) were cultured for 24 h in media supplemented with either 5 µM or 40 µM iron (FAC). β-actin was detected as loading controls. (F) Senescent MEFs possess enhanced resistance to elevated iron. The viability of iron treated primary and senescent MEFs was determined by propidium iodide and flow cytometry. Note that an extremely high concentration of iron (500 µM) was used as a positive control. (G) Chelation reduced intracellular iron and ferritin accumulation in irradiated MEFs. Primary MEFs were treated with the cell-permeable iron chelator deferiprone (Dfp) (50 µM) while irradiated (10 Gy) to become senescent and then subsequently for 10 days in culture. Primary MEFs irradiated and cultured in basal medium served as a control. (i) ICP-MS analyses demonstrated that sustained Dfp treatment prevented iron accumulation in irradiated MEFs. Baseline intracellular iron level in primary MEFs (MEF PRI) is also shown. (ii) Western blot analyses and densitometry demonstrated that sustained Dfp treatment prevented ferritin accumulation in irradiated MEFs. β-actin was detected as a loading control. (H) Percentage of senescent MEFs following sustained Dfp treatment. Primary MEFs were treated with Dfp (50 µM) while irradiated (10 Gy) to become senescent and then subsequently for 10 days in culture. Primary MEFs irradiated and cultured in basal medium served as a control. The majority (> 80%) of treated MEFs become senescent, staining positive for SA-βgal activity (blue staining).(I) Stored iron in senescent MEFs once accumulated is highly resistant to chelation. 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. (ii) Western blot analyses and densitometry showed a negligible effect of Dfp treatment on ferritin level of senescent MEFs. β-actin was detected as a loading control. Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
Fig. 5
Fig. 5
Iron accumulation in senescent MEFs is associated with impaired ferritinophagy. (A) Ferritin degradation in primary MEFs (MEF PRI) was inhibited by lysosomal acidification. (i) Primary MEFs (MEF PRI) were treated with 40 µM iron (FAC) for 24 h to increase ferritin expression. Ferritin degradation was stimulated with the iron chelator deferoxamine (Dfo) (50 µM) and occurred substantially with 6 h of treatment, as determined by western blot analyses (ii) ICP-MS analyses confirmed that intracellular iron levels increased in primary MEFs following treatment with 40 µM iron (FAC) for 24 h (PRI Fe), subsequently becoming comparable to iron levels observed in senescent MEFs (MEF IR) (14 days post-irradiation).(B) Ferritin degradation in senescent MEFs (MEF IR) is impaired. Western blot analysis showing no change to ferritin in senescent MEFs (MEF IR) (14 days post-irradiation) treated with Dfo (50 µM) for 6 h. β-actin was detected as a loading control.(C) Autophagy in primary MEFs with functional lysosomes can be monitored by the LC3-II: LC3-I ratio. The ratio of LC3-II: LC3-I was determined by western blot analyses and densitometry and increased in primary MEFs (MEF PRI) treated with the autophagy activator rapamycin (100 nM) for 16 h. The ratio of LC3-II: LC3-I was decreased in primary MEFs (MEF PRI) treated with the autophagy inhibitor bafilomycin A1 (100 nM) for 16 h. β-actin was detected as a loading control. (D) Senescent MEFs (10 days post-irradiation) displayed a large build-up of LC3-II and elevated levels of p62, indicating impaired lysosomal function, as determined by western blot analyses and densitometry. The levels of LC3-II protein and p62 in primary MEFs (MEF PRI) is shown for comparison. β-actin was detected as a loading control. (E) Senescent MEFs are highly resistant to ferroptosis. The viability of primary and senescent MEFs treated for 24 h with varying concentrations of erastin (0.1–5 μM) and RSL3 (0.1–5 μM) was determined by propidium iodide and flow cytometry. (F) Preservation of autophagy averted the accumulation of iron-regulatory proteins in irradiated MEFs. Primary MEFs were treated with rapamycin (100 nM) while irradiated (10 Gy) to become senescent and then subsequently for 10 days in culture. Western blot analyses demonstrated that sustained rapamycin treatment significantly reduced expression of phosphorylated ribosomal protein S6 (pS6). LC3-II levels were likewise significantly reduced indicating preservation of lysosomal function and permissible LC3-II degradation. Rapamycin treatment averted the iron accumulation phenotype of senescent MEFs, preventing the accumulation of TfR1 and ferritin (densitometry shown). β-actin was detected as a loading control. (G) Preservation of autophagy averted the iron accumulation in irradiated MEFs. Primary MEFs were treated with rapamycin (100 nM) while irradiated (10 Gy) to become senescent and then subsequently for 10 days in culture. Primary MEFs irradiated and cultured in basal medium served as a control. ICP-MS analyses demonstrated that sustained rapamycin treatment averted intracellular iron accumulation in irradiated MEFs. (H) Percentage of senescent MEFs following sustained rapamycin treatment. Primary MEFs were treated with rapamycin (100 nM) while irradiated (10 Gy) to become senescent and then subsequently for 10 days in culture. Primary MEFs irradiated and cultured in basal medium served as a control. Approximately 50% of treated MEFs became senescent, staining positive for SA-βgal activity as established by blue staining. Images were taken at 100× magnification. (I) Autophagy activation reduced stored iron in established senescent MEFs. ICP-MS analyses demonstrated that senescent MEFs (MEF IR) (10 days post-irradiation) treated for 4 days with rapamycin (100 nM) showed a significant reduction in intracellular iron when compared to untreated senescent MEFs (MEF IR). (J) Preservation of autophagy was insufficient to sensitize irradiated MEFs to ferroptosis. The viability of primary, senescent, and rapamycin treated irradiated MEFs (MEF IR + Rapa) (as described previously in Fig. 5G), treated for 24 h with varying concentrations of erastin and RSL3 (0.1–5 μM) was determined by propidium iodide and flow cytometry. Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
Fig. 6
Fig. 6
The enrichment of senescent cells in ageing mouse liver is accompanied by iron accumulation and elevated ferritin. (A) Senescent cells are markedly enriched in the livers of aged mice. Percentage of senescent cells in serial histological liver sections from both young (3 month; n = 4) and aged mice (30 month; n = 4) as determined by SA-βgal activity (blue staining). (B) ICP-MS analyses demonstrated a significant increase in iron (~ 2.5-fold) in liver tissues harvested from aged mice (30 month; n = 4) when compared to liver tissues harvested from young mice (3 month; n = 4). (C) The iron-storage protein ferritin and the senescence marker p16 were significantly elevated in livers of aged mice. (i) Western blot analyses and densitometry demonstrated that ferritin and p16 expression are increased in liver tissues harvested from aged mice (30 month; n = 3) when compared to liver tissues harvested from young mice (3 month; n = 3). β-actin was detected as a loading control. (ii) Quantitative real-time PCR analysis confirmed increased p16 transcript levels in liver tissues harvested from aged mice (30 months, n = 3), when compared to liver tissues from young mice (3 months, n = 3). (D) The percentage of ferritin-enriched cells within aged livers matched the percentage of SA-βgal positive senescent cells. Serial histological liver sections from both young (n = 3) and aged mice (n = 3) were stained for either SA-βgal activity or for ferritin. Images are representative of the mean percentage of either SA-βgal activity or ferritin staining from each mouse liver, as determined by observing 4 independent fields of view at a magnification of 400X using OlyVIA viewer software (ver2.4). Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
Fig. S1
Fig. S1
Relevant experimental controls for inducing cellular senescence. (A) Primary MEFs were exposed to a range of gamma irradiation (10–50 Gy) doses to induce senescence. Across all doses the majority (> 80%) of MEFs displayed positive SA-βgal activity at 10 days post-irradiation (blue staining). (B) The viability of MEFs exposed to a range of gamma irradiation (10–50 Gy) doses was determined by propidium iodide and flow cytometry at 10 days post-irradiation. There was no change in cell viability across the doses. (C) Real time qPCR analyses showed elevated mRNA transcripts for senescence-associated genes p16, p21 and IL-6 in senescent MEFs (MEF IR) at 21 days post-irradiation (10 Gy). Levels were normalised against GAPDH and expressed as fold change relative to primary MEFs (MEF PRI). (D) ICP-MS analyses demonstrated that MEFs exposed to a range of gamma radiation dose (10–50 Gy) all accumulated similar levels of elevated intracellular iron at 10 days post-irradiation. (E) Irradiated p53 mutant MEFs (IR p53 mut) displayed (i) significantly lower SA-βgal positivity (~ 20%) compared to wild-type MEFs (~ 80%) at 10 days post-irradiation (10 Gy) and (ii) had a negligible increase (< 2-fold) in intracellular iron, as determined by ICP-MS analyses. Non-irradiated p53 mutant MEFs (PRI p53 mut) had comparable intracellular iron levels when compared to non-irradiated p53 wild type MEFs (MEF PRI). Senescenct wild-type MEFs (MEF IR) are included for comparison. (F) MEFs induced to become senescent [irradiation (IR) or replicative exhaustion (REP)] have increased adhered cell diameter (Feret) (≥ 2 fold) compared to primary MEFs (MEF PRI), but comparable cell diameter in suspension following trypsinization. Cells were imaged using a bright field inverted microscope (Olympus IX51) and subsequently photographed (Canon 1100D). Feret diameter (caliper length) was determined using ImageJ software (n = 3). Suspended cell diameter (post-trypsinization) was determined using a Beckman Coulter Z Series Cell Count and Size Analyser (n = 3). (G) Cellular protein content as a marker for overall cell mass and volume. Irradiated (MEF IR), replicative (MEF REP) and oncogenic-induced (MEF OIS) senescent MEFs contained ~ 1.3-fold, ~ 1.1-fold and ~ 1.2-fold more protein in comparison to primary MEFs (MEF PRI), respectively. Cellular protein concentrations were determined using the BCA protein assay kit (Thermo Scientific) as per manufacturer’s instructions. Total protein was expressed as mg per 106 cells and represented as mean ± SD (n = 3). (H) Proliferating primary MEFs lose replicative potential as a result of continual passaging. The population-doubling limit per day (PDL/day) reached 0.2 at passage 6 prior to cell cycle arrest at passage 7 (senescence). Population doubling was determined by the following formula: n = 3.32 (log UCY − log l) + X, where n = the final PDL number at end of a given subculture, UCY = the cell yield at that point, l = the cell number used as inoculum to begin that subculture, and X = the doubling level of the inoculum used to initiate the subculture being quantitated. (I) Natural variance in intracellular iron concentration between primary MEFs derived from different embryos (same pregnancy) ICP-MS analyses of isogeneic C57BL/6 MEF lines (n = 10) showed a range of intracellular iron (84.2–181.9 ng/106 cells) concentrations. Data represented as mean ± range (n = 10). Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001).
Fig. S2
Fig. S2
Subcellular localization of ferroportin is altered in senescent MEFs. (A) Senescent MEFs at day 21 post-irradiation (MEF IR) displayed ferroportin predominately at an intracellular localization (indicated by arrows). Ferroportin was detected with primary anti-ferroportin antibody (Cat#NBP1-21502, Novus Biologicals) and secondary goat anti-rabbit IgG Alexa 488. Nuclear staining was performed using ethidium bromide. Ferroportin localization in primary MEFs (MEF PRI) is also shown. (B) Fractionation of ferroportin in cytosolic and NP-40 insoluble fractions of primary (MEF PRI) and senescent MEFs (MEF IR) at 21 days post-irradiation (10 Gy). Increased ferroportin in senescent MEFs was predominantly restricted to the insoluble fraction. (C) Ferroportin in plasma membrane enriched fractions of primary (MEF PRI) and senescent MEFs (MEF IR) at 21 days post-irradiation (10 Gy). In the plasma membrane fraction of senescent MEFs (MEF IR), ferroportin expression was significantly decreased, while TfR1 expression was increased, when compared to primary MEFs (MEF PRI).
Fig. S3
Fig. S3
Iron accumulation in replicative senescent MEFs is maintained and associated with impaired ferritinophagy. (A) Intracellular iron accumulation occurred concurrent with replicative senescence in passaging MEFs (MEF REP). Percentage of senescent MEFs in passaged cultures (passages 4, 6 and 7) as determined by SA-βgal activity (blue staining). Images were taken at 100X magnification. (B) ICP-MS analyses demonstrated that iron accumulated when replicative senescence commenced ( < 10%) at passage 6 and was augmented with senescence enrichment (>80%) at passage 7. Note that iron accumulation somewhat plateaued (~18-fold) after culturing passage 7 senescent MEFs for 10 days. (C) Western blot analyses demonstrated that replicative senescent MEFs (MEF REP) have increased TfR1, ferritin and LC3-II in comparison to primary MEFs (MEF PRI). The large build-up of LC3-II indicates impaired lysosomal function. β-actin was detected as a loading control. Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
Fig. S4
Fig. S4
Inhibiting mTOR in senescent cells prevented iron accumulation. (A) Inhibiting mTOR does not cause escape from cell cycle arrest. Primary MEFs were treated with rapamycin (100 nM) while irradiated (10 Gy) to induce senescence and then subsequently for 10 days in culture. Treated MEFs were unable to proliferate when seeded in 6 well plates (100,000 cells/well) and cultured for additional 5 days. (B) Inhibiting mTOR in senescent MEFs after 10 days post-irradiation through rapamycin (100 nM) treatment for 4 days, had no effect on SA-βgal activity (>80%) (blue staining). (C) Primary MEFs were treated with torin 1 (50 nM) while irradiated (10 Gy) to induce senescence and then subsequently for 14 days in culture. Sustained torin 1 treatment significantly prevented intracellular iron accumulation, as determined by ICP-MS. Statistical analysis was performed by student-t-test: significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data represented as mean ± SD (n = 3).
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