Fig. 2: ISG15 expression is linked to mitochondria-related pathways.
From: ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity
a Gene sets enriched in the transcriptional profile of tumors belonging to the top ISG15 high-expression group, compared with the bottom expression group in the Bailey data series. Shown are the NES (normalized enrichment score) values for each pathway using the Hallmark genesets, meeting the significance criteria: nominal p-value of <0.05, FDR < 25%. b Enrichment plot for OXPHOS signaling in ISG15 high versus low. c Autofluorescence (CSC) and Mitotracker Deep Red (MTDR, approximation of mitochondrial mass) were combined to sort the four gated populations (Q1–Q4) from Panc185 spheres. d WB analysis of ISG15 protein expression in the four FACS sorted populations in c. Tubulin was included as a loading control. e RTqPCR analysis of CMYC, KLF4 and ISG15 gene mean fold change ± sd in the four FACS sorted populations in c (n = 3 replicates from one independent sorting experiment). Values compared to Q1, set as 1.0. f WB analysis of ISG15 protein expression in mitochondrial and cytosolic fraction from Panc185 Control or CRISPR-Cas9 ISG15-edited (ISG15CRISPR) CSCs (sph) and non-CSCs (adh). The membrane was additionally blotted for mitochondria OXPHOS complex proteins using the Mitoprofile Total OXPHOS antibody cocktail in addition to GAPDH (loading control). Shown are bands corresponding to Complex (C)V, CIII, CII, CIV, and CI. Total ISG15 expression [ISG15-conjugated and monomeric (mon-) proteins] for each sample was quantified by densitometric analysis and fold changes are shown, relative to control adherent (adh), set as 1.0 for both mitochondria and cytosolic fractions.
