Fig. 4: Loss of ISG15/ISGylation alters mitochondrial state and metabolism.

a Transmission electron micrographs of control and ISG15^CRISPR Panc185 cells. Scale bars = 2 µM. b RTqPCR analysis of fold-change in mitochondrial DNA (mtDNA) gene 12s mean copies ± sd in control and ISG15^CRISPR Panc185 and Panc354 cells. Data are normalized to β-Actin expression and control set as 1.0. (n = 3 biologically independent samples; ***p < 0.0001, as determined by Student’s t-test). c, d Representative histograms of flow cytometric analysis of NAO (10-N-Nonyl acridine orange) in control and ISG15^CRISPR Panc185 and Panc354 cells c, and mean percentages ± sd in mitochondrial mass as a function of Mitotracker Green (MTR-G) and NAO (10-N-Nonyl acridine orange) staining d (Panc185: MTR-G n = 4 biologically independent samples **p = 0.0092 and NAO n = 8 biologically independent samples ***p < 0.001, as determined by Student’s t-test; and Panc354: MTR-G and NAO n = 3 biologically independent samples ***p < 0.001, as determined by Student’s t-test). e Representative histograms of Mitotracker CM-H₂XRos in control and ISG15^CRISPR Panc185 and Panc354 cells. f Representative plot of mean Oxygen Consumption Rate (OCR) levels ± sem, normalized to protein content, for control and ISG15^CRISPR Panc185 cells, which were treated with O (oligomycin), F (FCCP), A (antimycinA) and R (rotenone) into culture medium. Continuous OCR values (pmoles/min/µg protein) are shown. g Measured and mean calculated parameters of OCR ± sem (n = 6 measurements per time point examined over five independent experiments; *p = 0.0148; **p = 0.0088; ***p < 0.001; *p = 0.0366; ns, not significant, as determined by Student’s t-test).