Accelerated replication fork progression in cells expressing high levels of ISG15 is largely conjugation independent. (A) ISG15 immunoblot of U2OS FIT cells bearing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after 48-h induction with 1 µg/ml doxycycline (dox). (B) The effect of ISG15 on DNA replication was assessed by using DNA fiber assay in parental U2OS FIT cells (as in A) and U2OS FIT cells (ISG15 KO [U2OS ISG15/KO]; see Fig. S3 A) expressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h). At least 100 tracks were scored per sample (n = 3). Vertical lines represent the median value, and boxes and whiskers show 10–90th percentiles. Statistical analysis according to Mann–Whitney test; ns, not significant; ****, P < 0.0001; ***, P < 0.001. (C) Colocalization of FLAG-ISG15 and FLAG-ISG15ΔGG with PCNA (FLAG/PCNA) in U2OS FIT cells induced for 48 h with 1 µg/ml doxycycline determined by PLA. QIBC shows the distribution of PLA foci counts. For each condition, images containing ≥1,000 cells per experiment were acquired (n = 3). (D) Representation of 3D structure of N-terminal ubiquitin-like domain (N-lobe) of ISG15 and ubiquitin (Protein Data Bank accession nos. 1Z2M and 1UBQ, respectively). The residues corresponding to the hydrophobic patches are indicated. (E) U2OS FIT cells were transfected with EV, FLAG-ISG15 wild type and ISG15 mutants carrying the indicated single amino acid substitutions (L10A, L72A, and V74A) or the combination of them (LLVAAA). (F) Analysis of IdU track length measurements as in E. At least 100 tracks were scored per sample (n = 3). Vertical lines represent the median value, and boxes and whiskers show 10–90th percentiles. Statistical analysis according to Mann–Whitney test; ns, not significant; ****, P < 0.0001.