Fig 2: CD95-induced senescence is mediated by low-level canonical caspase signaling. (a) CRC29 colonospheres were exposed once (cycle 1) or chronically exposed to CD95L (10 ng/ml) for 2 weeks (cycle 2), and collected at the indicated time points. Cells were collected and analyzed for the presence of activated (cleaved) caspase-3 by western blotting. (b–c) CRC29 colonospheres were exposed to CD95L (5 ng/ml) in the presence or absence of zVAD (25 μM) for 1 or 7 days and were then processed for imunofluorescence analysis of caspase-3 activation (b) and DNA damage (γ-H2AX) (c) An overview of the significance of all comparisons is provided in Supplementary Table S1. (d) Colonspheres were exposed to CD95L (10 ng/ml) for 24 h in the presence or absence of zVAD (25 μM) followed by western blot analysis of caspase cleavage and iCAD processing. (e and f) CRC29 colonospheres were exposed to increasing concentrations of CD95L as indicated in the presence or absence of zVAD (25 μM), after which SA-βGAL (e) and clone-forming capacity (f) were assessed at 7 or 14 days, respectively. The asterisks indicate significant differences (ordinary one-way ANOVA) (****P<0.00001; **P<0.001). An overview of the significance of all comparisons is provided in Supplementary Table S1

Fig 3: ASIC1a suppresses the growth and proliferation of glioma cells through the induction of apoptosis. (A-C) Apoptosis was analyzed by flow cytometric analysis of Annexin V-PE staining after shASIC1 treatment in (A, upper panel) glioma cells A172, (B, upper panel) U87MG and (C, upper panel) PDX-L12 cells. Apoptosis was also examined by overexpression of ASIC1a in (A, lower panel) A172, (B, lower panel) U87MG and (C, lower panel) PDX-L12 cells. (D-F) The cell cycle regulatory proteins cyclin D1 and p21, as well as apoptotic-related protein Fas were examined when the ASIC1 gene was silenced by shASIC1a in (D, upper panel) A172, (E, upper panel) U87MG, and (F, upper panel) PDX-L12 cells. Cyclin D1, p21, and Fas were also detected when the ASIC1a gene was overexpressed in (D, lower panel) A172, (E, lower panel) U87MG and (F, lower panel) PDX-L12 cells. The bar graphs indicate the mean ± SD of three independent experiments. All data represent a representative experiment from three independent experiments. *P<0.05 and **P<0.01. ASIC1a, acid-sensing ion channel 1a; sh, short hairpin.

Fig 4: CD95 stimulation causes reduced proliferation and induction of p53. (a) Bright field images of CR16 cells (upper panel) show the increase in cell size (indicated by white arrows) following 1-week treatment with FC or FC-CD95L. Immunofluorescence analysis (lower panel) for Ki67 (red), ß-catenin (green) and nuclei (DAPI, blue) of control and CD95L-treated CRC29 colonospheres. Cells were chronically treated for 2 weeks, plated as single cells in Matrigel in the absence or presence of CD95L for 7 days. (b) Quantification of the number of cells with aberrantly shaped or multiple nuclei in control and CD95L-treated CRC29 cultures treated as in (a). (c) Quantification of the number of Ki67-positive cells in control and CD95L-stimulated CRC29 colonospheres treated as in (a). (d) Immunofluorescence analysis of p53 and p21 induction in CRC29 colonospheres treated for 24 h with FC control and FC-CD95L. ***P<0.00001

Fig 5: CD95-indcued senescence depends on p53. (a) CRC29 colonospheres were transduced with a lentiviral vector expressing control shRNA or shRNA-targeting p53, and were subsequently exposed to FC or FC-CD95L. CD95L-induced activation of caspase-8, caspase-3, cleaved iCAD, DNA damage (?-H2AX), and p21 induction were then assessed by western blotting. (b and c) Control CRC29 and two independent clones of p53-suppressed colonospheres were exposed to FC-CD95L for 14 days, and analyzed for SA-ßGAL activity (b) and for clone-forming capacity (c). The asterisks indicate significant differences (ordinary one-way ANOVA) (****P<0.00001; **P<0.001; *P<0.01). An overview of the significance of all comparisons is provided in Supplementary Table S1