Fig. 5: Loss of ISG15/ISGylation reduces mitophagy.
From: ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity
a Transmission electron micrographs of control and ISG15CRISPR Panc354 cells. Scale bars = 1 or 2 µM. b Immunofluorescent images of TOM-20 in control and ISG15CRISPR Panc354 cells. Nuclei stained with DAPI in blue. Scale bars = 10 µM. c Mitochondrial network from b displayed as a skeleton network using ImageJ skeleton filter. Scale bar = 10 µM. d Representative histograms of MTDR expression in control and ISG15CRISPR Panc185 and Panc354 cells after 6 h of treatment with CsA (5 µM) or without (Ctl). e Mean fold change in MTDR levels ± sd in control (Ctl)- and CsA-treated cultures (left) and mitophagic flux determined as the ratio of MTDR accumulation after CsA treatment compared to its respective control (right) in control and ISG15CRISPR Panc185 and Panc354 cells (Panc185: n = 4 biologically independent samples; **p = 0.0013; *p = 0.0191; *p = 0.0111, as determined by Student’s t-test; and Panc354: n = 4 biologically independent samples; ***p < 0.001; ns, not significant; **p = 0.0041, as determined by Student’s t-test). f Mean fold change in Keima protein fluorescence levels ± sem in control (red) and ISG15CRISPR (blue) Panc185 and Panc354 cells. Cells were untreated or treated with FCCP (16 µM), with or without co-treatment with the autophagy inhibitors CsA (5 µM), Bafilomycin (Baf; 150 nM) or Chloroquine (Chlq; 50 µM). Fluorescence in control-treated cells is set to 1.0. (n = 4 biologically independent samples for Panc185 and n = 3 biologically independent samples for Panc354; **p < 0.01; ***p < 0.001, ns, not significant, as determined by one-way ANOVA with Dunnett’s multiple comparison test). g Summary of average fold change in parkin levels ± sem, determined by flow cytometric analysis, in control and ISG15CRISPR Panc185 and Panc354 cells untreated (Ctl) or treated with FCCP (16 µM) plus CsA (5 µM) for 4 h (Panc185: n = 4 biologically independent samples; *p = 0.0283; **p = 0.0013, as determined by Student’s t-test; and Panc354: n = 4 biologically independent samples **p = 0.0084; ns, not significant, as determined by Student’s t-test).
