Research Paper Volume 9, Issue 10 pp 2137—2162

Figure 6. Ferroptosis sensitivity in senescent cells depends on the trigger. (A) Senescence was assessed by staining cells for the Senescence-Associated β-Galactosidase in IMR90 cells expressing either a control vector (V), the RASV12 oncogene (RAS), PML or STAT5A (cS5A). (B) Immunofluorescence of SOCS1 (anti-SOCS1) and phosphorylated ATM at S1981 (anti-ATM) in IMR90 cells rendered senescent by overexpressing the RASV12 oncogene compared to IMR90 expressing a control vector (Vector). (C) QPCR for mRNA levels of SOCS1-dependent p53 target genes in IMR90 cells expressing a control vector (V) or rendered senescent by overexpression of RASV12 (RAS) or PML (PML). (D) IC50 curves of IMR90 cells overexpressing a control vector (V), the RASV12 oncogene (RAS), PML or STAT5A (cS5A). Cells were treated 24 hours after plating with 12 different doses (0, 10, 20, 40, 60, 80, 100, 120, 160, 180 and 200 µM) of tert-butyl-hydroperoxide (TBH). Cells were fixed and stained with Crystal Violet to assess cell death 16 hours after treatment. The dye was then solubilized with acetic acid 10% and measured with a spectrophotometer. (E) The value of IC50 of each condition graphed in D is presented. No IC50 could be calculated for PML as it was resistant at the doses used. (F) GSH quantification in IMR90 cells rendered senescent by overexpression of RASV12, PML IV or STAT5A, compared with empty vector control (V). All experiments were performed three times, error bars indicate the standard deviation of triplicates, * = p<0.05, using the Student’s t test, **=p<0.01, ***=p<0.005.