Fig 1: Drp1 and Bim mediate AICD progression downstream of JNK1 and ERK1/2.Jurkat cells have been transfected with the indicated plasmids (pEYFP-Drp1-S616E and Flag-Bim-L). 24 h after transfection, Jurkat cells have been stimulated for AICD in presence or not of FR180204, as indicated. a Relative viability (AICD:unstimulated ratio) of YFP-Drp1 and Bim-L transfected Jurkat cells after 32 h from AICD induction (percentage of annexinVneg7AADneg cells assessed by flow cytometry). Cells have been gated on YFP-positive cells when transfected with YFP-Drp1-expressing plasmid (n = 5). b Representative single z-stack images of mitochondria morphology (TOM20) and cytochrome-C (light blue) 28 h after AICD induction in Jurkat cells transfected with YFP-Drp1 and Bim-L. Quantification of the percentage of cells with fully released cyt-C is reported in the graph below (n = 6). c Expression levels of the indicated (phospho)-proteins 28 h after AICD induction in Jurkat cells transfected with YFP-Drp1 and Bim-L. Opa1, Hsp90, TOM20 and GAPDH have been evaluated in samples treated with BMH (+BMH) to isolate Opa1 oligomers, which are indicated with an asterisk (*). Quantifications of the amount of s-OMA1 and Opa1 oligomers are reported in the graph below (n = 5). One-way ANOVA have been performed to analyze s-OMA1 levels. One-way ANOVA on repeated measurements have been performed to analyze Opa-1 oligomers. d Representative electron micrographs of Jurkat cells transfected with YFP-Drp1 and Bim-L 28 h after AICD induction. Mitochondria have been categorized according to cristae alterations (normal, partially or completely altered) and the quantification of the (AICD - unstimulated) difference in the percentage of mitochondria belonging to the completely altered-cristae category is reported in the graph below (n = 3). e. Representative images of mitochondria morphology (TOM20, confocal z-stack reconstructions) 28 h after AICD induction in Jurkat cells transfected with YFP-Drp1 and Bim-L. Quantification of the percentage of cells with fragmented mitochondria in each condition is reported in the graph on the right (unstim: n = 3; AICD: n = 5). f TMRE profile by flow cytometry in Jurkat cells transfected with YFP-Drp1 and Bim-L 28 h after AICD induction in presence or not of FR180204. Percentage of cells with polarized mitochondria are quantified in the graph on the right (n = 7). Cells have been gated on YFP-positive cells when transfected with YFP-Drp1-expressing plasmid. Data are shown as mean ± SEM. Scale bar, 10 µm in b, e, 0.4 µm in d. Significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.