Increase of replication fork progression by ISG15 depends on the functional interaction with RECQ1. (A) Chromatin extracts were obtained from U2OS FIT cells expressing EV or FLAG-ISG15ΔGG and processed as indicated for mass spectrometry analysis. (B) ISG15 interaction with RECQ1 was measured as luminescence signal by NanoBRET in HEK293T cells that were cotransfected with HaloTag-Fusion constructs of ISG15 (HT-ISG15) and RECQ1 fused to NanoLuc at N-terminus (NL-RECQ1) or C-terminus (RECQ1-NL; n = 3). HT-p53 and NL-MDM2 are used as controls. (C) Representative images of colocalization of FLAG-ISG15 and FLAG-ISG15ΔGG with endogenous RECQ1 (ISG15/RECQ1) in U2OS FIT cells after doxycycline induction (1 µg/ml, 48 h), assessed by PLA. Scale bars, 10 µm. (D) QIBC shows the distribution of PLA foci counts of samples described in C. For each condition, images containing ≥1,000 cells per experiment were acquired (n = 3). (E) FLAG-ISG15 and RECQ1 protein levels in U2OS FIT cells expressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h) and upon knockdown of RECQ1 by siRNA transfection. siLuc is used as control. (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. (G) Left: DNA fiber–labeling strategy to determine fork restart and representative images. Right: Fork restart speed (IdU) normalized on fork speed (CldU) in U2OS FIT cells upon RECQ1 knockdown after doxycycline induction (1 µg/ml, 48 h) as described in the scheme. At least 100 tracks were scored per sample (n = 3). Statistical analysis according to Mann–Whitney test; ns, not significant; ****, P < 0.0001. (H) Percentage of stalled forks after HU washout in U2OS FIT cells described in G.