Fig. 2: Treatment with SP600125 and FR180204 inhibitors prevents mitochondrial alterations upon AICD in hPBT cells.

a–e hPBT cells have been stimulated for AICD induction in presence or not of SP600125. In a is reported the relative viability (AICD:unstimulated ratio) assessed by flow cytometry (percentage of annexinV^negative7AAD^negative cells assessed by flow cytometry) (n = 5). In b are reported representative confocal z-stack reconstructions of mitochondria (TOM20) in hPBT cells 2 h after AICD induction (n = 4). In c are reported representative images of viable and dying hPBT cells showing mitochondria (TOM20 in green, confocal z-stack reconstruction) and cytochrome-C (red) localization, 4 h after AICD induction. Quantification of the cyt-C co-localization index with mitochondria (see Methods for details) in presence or not of SP600125 4 h after AICD induction is reported in the graph on the right (n = 4). In d is reported the TMRE profile by flow cytometry 2 h after AICD induction. Percentages of cells with polarized mitochondria (indicated with red bars on plots) are quantified in the graph on the right (n = 3). In e are reported representative electron micrographs of hPBT cells 4 h after AICD induction. Quantification of the fraction of mitochondria with altered cristae per cell (range 0–1) in each condition is reported in the graph on the right (at least 30 cells per condition from n = 3 independent experiments). f Relative viability (AICD:unstimulated ratio) of hPBT cells stimulated for AICD induction in presence or not of FR180204 (percentage of annexinV^negative7AAD^negative cells assessed by flow cytometry) (n = 4). g Representative confocal z-stack reconstructions of mitochondria (TOM20) in hPBT cells 2 h after AICD induction in presence or not of FR180204. Quantification of the cells with fragmented mitochondria is reported in the graph on the right (n = 4). h Representative images showing mitochondria (TOM20 in green, confocal z-stack reconstructions) and cytochrome-C (red) localization 4 h after AICD induction in hPBT cells, in presence or not of FR180204. Quantification of the cytochrome-C co-localization index with mitochondria (see Methods for details) is reported in the graph on the right (n = 4). i Expression levels of the indicated (phospho)-proteins in hPBT cells 2 h after AICD induction, in presence or not of SP600125. Relative quantification of the AICD:unstimulated ratio for each protein is reported in the graph on the right (n = 4). j Jurkat cells have been transfected with pEYFP-C1-Drp1 and/or Flag-MKK7B2JNK1a1 plasmids. After 18 h, cells were incubated or not in presence of SP600125 for 6 h and then analyzed for the expression levels of the indicated (phospho)-proteins (one experiment representative of three independent experiments). k, l HeLa cells were transfected or not with Flag-MKK7B2JNK1a1 plasmid. After 24 h, cells were lysed and protein extract immunoprecipitated (IP) using anti-Drp1, anti-Flag or control IgG antibody. In k is reported the co-immunoprecipitation between Drp1 and MKK7B2JNK1a1 in anti-Flag immuno-precipitated lysates. In l an in vitro kinase assay has been performed with IP-purified endogenous Drp1 (from untransfected HeLa) and Flag-MKK7B2JNK1a1 (from HeLa cells transfected with Flag-MKK7B2JNK1a1) in presence or not of SP600125 for 30 min; then the levels of the indicated (phospho)-proteins has been measured by western blot. All experiments are representative of three independent experiments. m Expression levels of the indicated (phospho)-proteins in hPBT cells 2 h after AICD induction in presence or not of SP600125. Relative quantification of the AICD:unstimulated ratio for each Bim isoform is reported in the graph below (n = 5). n Expression levels of the indicated (phospho)-proteins in hPBT cells 2 h after AICD induction in presence or not of FR180204. Relative quantification of the AICD:unstimulated ratio for each protein is reported in the graph on the right (Drp1: n = 3; Bim n = 5). Data are shown as mean ± SEM. Scale bar, 5 μm in b, c, g, h, 0.15 μm in e. Significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001.