Fig. 8

Ru1 affects PaCSC oxygen consumption and mitochondrial functional properties. A Representative plot showing mean ± sd of oxygen consumption rate (OCR) for untreated (CTL) and Ru1-treated Panc185 spheres (100µM, 24 h), normalized to total protein using a BCA kit, which were treated with distinct inhibitors of mitochondrial function: O (oligomycin), F (FCCP), A (antimycinA), and R (rotenone). Continuous OCR values (pmoles/min/no. cell) are shown. B Measured and calculated mean ± SD OCR parameters (Resp = Respiration; Max = Maximum; SRC = Spare Respiratory Capacity; OC = Oxygen consumption; n=3 biological replicates with 3 readings). ** p < 0.01, *** p < 0.001, ns = not significant, as determined by unpaired two-sided Student’s t-test. C Mean fold-change ± SD in ATP nmoles/mg protein in untreated (CTL) and Ru1-treated Panc185 and PancA6L (100µM, 24 h) cells compared to control, set as 1.0. *** p < 0.001, as determined by unpaired Student’s t test. D Mean fold-change ± SD in the mitochondria membrane potential probes CMX-ROS, CM-H2Xros and Mitoblue or the ROS probe MitoSOX as a function of increasing concentrations of Ru1 in Panc185 or PancA6L cells (48h). * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by one-way ANOVA with Dunnett post-test, compared to untreated (Ctl) set as 1.0. E Representative IF confocal images of TMRE (mitochondria membrane potential) or CellROX DeepRed (ROS) staining in untreated (Control), Ru1 or Ru1-met-treated Panc185 cells (100µM, 24 h). F Mean fold-change ± SD in the mitochondria membrane potential probe CMX-ROS or the ROS probe MitoSOX in Autofluorescent-negative (Fluo-) or Fluo+ FACS-sorted cells pre-treated with Ru1 (100µM, 48h). * p < 0.05, ns = not significant, as determined by unpaired Student’s t test. G Mean fold-change ± SD in the expression of the mtDNA-encoded gene MTATP6 or the nuclear DNA encoded gene COX5 as a function of increasing concentrations of Ru1 in Panc185 or PancA6L cells (48h treatment). Values were normalized to ß-actin levels. *** p < 0.001, **** p < 0.0001, as determined by one-way ANOVA with Dunnett post-test, compared to untreated (Ctl)