Fig. 5: mPTP accounts for cell death sensitivity in MTFP1 deficient cells.

a Representative confocal images of WT and Mtfp1^−/− MEFs subjected to H₂O₂ treatment. The pan-caspase and cyclophilin D inhibitors, q-VD-OPh hydrate (qVD) and cyclosporin A (CsA) respectively, were used to block both caspase and mPTP dependent cell death. Cell death was monitored by Propidium Iodide uptake (PI, orange) and imaging cells every hour (h) for 18 h. Scale bar = 100 μm. b Kinetics of PI uptake was determined by counting the number of PI⁺ positive cells (orange) over total number cells nuclear stained with NucBlue (NB, blue) and expressed as % PI⁺/NucBlue. Data are means ± SD of n = 6 independent experiments. c one-way ANOVA of b at 18 h, *p < 0.05. d Validation of Cyclophilin D (CYPD) ablation by Crispr/Cas9 genome editing of Ppif in WT and Mtfp1^−/− MEFs. Equal amounts of protein extracted from WT, Mtfp1^−/−, Ppif^−/−, Mtfp1^−/− Ppif^−/− MEFs (n = 3) were separated by SDS–PAGE and immunoblotted with the indicated antibodies. SDHA was used as mitochondrial marker and loading control. e Representative confocal images of WT, Mtfp1^−/−, Ppif^−/−, Mtfp1^−/− Ppif^−/− MEFs subjected to H₂O₂ treatment. Cell death was monitored by Propidium Iodide uptake (PI, orange) and imaging cells every hour (h) for 18 h. Scale bar = 100 μm. f Kinetics of PI uptake was determined by counting the number of PI⁺ positive cells (orange) over total number cells nuclear stained with NucBlue (NB, blue) and expressed as % PI⁺/NucBlue. Data are means ± SD of n = 3 independent experiments. g one-way ANOVA of f at 18 h; **p < 0.01, ****p < 0.0001. h Representative confocal images of WT, Mtfp1^−/−, Ppif^−/−, Mtfp1^−/− Ppif^−/− MEFs subjected to doxorubicin (DOXO) treatment. Live induction of the caspase 3/7 activation was monitored by using the CellEvent (CE, green). CellEvent positive cells (CE⁺, green) over total number cells NucBlue labeled (blue) were imaged every hour (h) for 18 h. Scale bar = 100 μm. i Kinetics of caspase 3/7 activation was determined by counting the number of CE⁺ positive cells (green) over total number cells nuclear stained with NucBlue (NB, blue) and expressed as % CE⁺/NucBlue. Data are means ± SD and representative of n = 3 independent experiments. j one-way ANOVA of i at 16 h, ****p < 0.0001.