Fig. 7: Loss of ISG15/ISGylation prevents PaCSC metabolic plasticity.

a Quantification of the average percentage ± sd of cells in early apoptosis (Annexin-V + ), late apoptosis (Annexin-V + /DAPI + ) and dead (DAPI + ) from control and ISG15^CRISPR sphere cultures after 3 days of treatment with Bafilomycin (1 nM, Baf, B) and/or Metformin (10 µM, MET, M) (n = 3 biologically independent samples; *p < 0.05; **p < 0.01; ***p < 0.001, as determined by Student’s t-test). b Experimental set-up for in vivo experiments using subcutaneously implanted control and ISG15^CRISPR tumors (xenografts) and treatment with Metformin (MET). c WB analysis of mon-ISG15 and ISG15-conjugated proteins in freshly digested donor tumors obtained from control and ISG15^CRISPR injected cells, used for tumor implantations in b. d Average fold change in tumor volume ± sem in control and ISG15^CRISPR xenografts with or without Metformin (MET) treatment over the course of 42 days (n = 6 tumors/group examined over two independent in vivo animal experiments; ***p < 0.001; ns, not significant, as determined by Student’s t-test). e Average tumor weights ± sem obtained at sacrifice from d (**p = 0.0099; ns, not significant, as determined by Student’s t-test). f Representative images of tumors at the end of the experiment.