Verifying cell responses to inhibitors. (A) Verification of whether a short or long time point of 20 µm Y-27632 treatment results in bioenergetic changes in Vcl KO cells using TMRM as a metric for mitochondrial membrane potential. (B) Fold change of TMRM signal of the cells treated with 20 µm Y-27632 relative to the vehicle control (deionized water) condition at each time point (N = 3, n = 70–85 cells). (C) Representative images of Vcl KO cells treated with 20 µm Y-27632 or the vehicle control for short and long time points. Scale bar, 100 µm. (D) Verification of whether a short or long time point of 25 µm Blebb treatment results in bioenergetic changes in Vcl KO cells using TMRM as a metric for mitochondrial membrane potential. (E) Fold change of TMRM signal of the cells treated with 25 µm Blebb relative to the vehicle control (DMSO) condition at each time point (N = 3, n = 53–62 cells). (F) Representative images of Vcl KO cells treated with 25 µm Blebb or the vehicle control for short and long time points. Scale bar, 100 µm. (G and H) Verification that cell treatment with (G) 20 µm oligomycin (N = 3, n = 90–96 cells) or (H) 10 µm iodoacetate (N = 3, n = 86 cells) treatments induced decreases in cell energy metrics. Bar graphs denote the mean ± SEM. ns = not significant, *P <0.05, **P < 0.01, ****P < 0.0001.