Fig 1: Increased cellular Ca2+ influx underlies mitochondrial failure and augmented senescence. a Changes in cytosolic-free Ca2+ concentration were analyzed in fura-2-loaded cells. Cells in HBSS containing 1.26 mM Ca2+ were subjected to 1-min depolarization with 90 mM KCl (red line). CaCl2 in the HBSS was increased to 5 mM during depolarization to facilitate the Ca2+ influx recording. In parallel experiments, 10 μM nifedipine was added to the assay medium during the recording (black line). Right panel: the increase of the F340/F380 ratio triggered by 90 mM KCl in the presence of VOCCs blockers is shown as mean ± s.d. of 3 experiments (a minimum of 70 cells per experimental condition). Final concentrations: 10 μM nifedipine, 1 μM ω-conotoxin MVIIC, 3 μM ML 218. b STIM1-KO cells, or STIM1-KO cells stably expressing a specific shRNA to knock-down CACNA1C transcripts, were treated as described in panel (a). The left panel shows a representative experiment, and the bar chart of the right panel shows the increase in the F340/F380 ratio evoked by depolarization (mean ± s.d. of two independent experiments; n > 60 cells per condition). c Senescence (left panel) and mitochondrial polarization (middle and right panels) were assessed from differentiated cells after 6 DIV, staining with C12FDG as described in Fig. 5c and TMRM as in Fig. 6b–d, respectively. Data are mean ± s.d. of three independent experiments (number of replicates is shown for each condition). d Rotenone-sensitive NADH oxidase activity was assessed from differentiated SH-SY5Y cell lysates (wild-type, STIM1-KO, and STIM1-KO + shRNA for CACNA1C). Data are presented as the mean ± s.d. of two independent experiments. e Cell were transiently transfected for the expression of the Ca2+ sensor 4mtD3cpv. Mitochondrial [Ca2+] was assessed as described in Fig. 7. Data of six independent experiments are shown in the right panel bar chart as mean ± s.d.