Fig. 5

PD-1 stimulation reduces the number of mitochondria but does not affect mitochondrial dynamics. a Number of mitochondria per cell as determined by direct counting from transmission electron microscopy images (n ≥ 83 cells/condition). Results are the average of counting by two independent observers, one of them blind to the experiment. b Relative mitochondrial DNA quantity determined by qPCR (n = 3). c Representative histogram of T_CTRL, T_ACT, and T_{ACT + PD1}-stimulated cells (48 h) stained with the MitoTracker Green probe. d Quantification of mean fluorescence intensity from cells as in c (n = 7 donors). e Quantification of MitoTrackerGreen mean fluorescence intensity in T_ACT and pre-treated T_{ACT + PD1} cells restimulated with T_CTRLand T_ACT beads (n = 3). f Representative confocal images of T_CTRL, T_ACT, and T_{ACT + PD1} cells stained with aconitase-2. g Quantification of mitochondrial circularity, determined from confocal images as in e using ImageJ software (n ≥ 31 cells/condition). h Quantification of OPA-1 and DRP-1 mRNA levels in T_ACT and T_{ACT + PD1}-stimulated cells. Values were normalized to those from T_CTRL cells. i Representative OPA-1 and DRP-1 immunoblots in cells treated as indicated. The line indicates removal of an empty lane. j Densitometric analysis of immunoblots as in h. The Rq was calculated as the ratio between each protein and β-actin, taking the value for T_CTRL cells as reference (n = 3 donors). In all cases, data were compared using one-way (a, b, g), two-way ANOVA (d, h, j) with Bonferroni’s post-test, or paired two-tailed Student’s t-test (e); * p < 0.05, ** p < 0.01, n.s., not significant