ISG15 expression levels impact replication fork progression in different systems. (A) Analysis of ISG15 protein levels in 12 of the 41 single clones tested, obtained from the CRISPR/Cas9 KO in MCF7 cells. Clone 1E was selected and used for the following experiments (in the main text referred to as MCF7 ISG15/KO). (B) ISG15 immunoblot on different cellular system/conditions used in our experiments and several cancer cell lines. HeLa were derived from cervical carcinoma; LS174T, SW620, and HT-29 were derived from colon cancer; M059K and T98G were derived from glioblastoma; DU145 and LNCaP were derived from prostate cancer. (C) Test of ISG15 knockdown efficiency in different cancer cell lines. Cells were transfected with siISG15 or siLuc as a control and collected after 24 h, and ISG15 levels were evaluated by Western blot analysis.