Accelerated replication fork progression in cells expressing high levels of ISG15 is largely conjugation independent. (A) ISG15 immunoblot on U2OS FIT cells expressing EV or FLAG-ISG15 and in U2OS FIT cells lacking the endogenous ISG15 (U2OS ISG15/KO) and reexpressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h). (B) Quantification of EdU incorporation in S phase of U2OS FIT cells expressing EV or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h) and grown in the presence of EdU (10 µM for 30 min) before collecting and processing for FACS analysis. Similar results were obtained in at least one independent experiment. (C) Fork asymmetry analysis in U2OS FIT expressing EV or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h). Each fork is described by the length of left and right IdU tracks. Red lines define a range of 30% difference between left and right tracks; left > right + 30% and right > left + 30% are considered asymmetric. Similar results were obtained in at least one independent experiment. (D) Left: Cell cycle profile of U2OS FIT cells expressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after 48-h induction with doxycycline. Right: Percentage of cell distribution in different cell cycle phases. (E) Sequence alignment of human ISG15 and ubiquitin (UB). The N- and C-lobes are indicated by bars. The conserved residues are in red. The residues that are part of the hydrophobic patch are indicated by asterisks (*). (F) Localization of ISG15 in U2OS FIT cells after doxycycline induction (1 µg/ml, 48 h). ISG15 immunostaining was performed after preextraction and MeOH fixation. Scale bars, 10 µm. (G) The indicated forms of ISG15 were expressed in HEK293T cells with or without the ISGylation machinery components UBE1L (E1), UBCH8 (E2), and HERC5 (E3). After 48 h, cell extracts were collected for Western blot analysis with the indicated antibodies.