Figure 1. ISG15 localizes at the DNA... | Rockefeller University Press

Figure 1.

$ISG15 localizes at the DNA replication forks and accelerates replication fork progression.(A) ISG15 immunoblot on protein extracts of U2OS FIT cells bearing EV or FLAG-ISG15, induced with doxycycline (dox; 1 µg/ml) for 48 h, and in parental U2OS treated with IFN-β (30 U/ml for 2 h) and chased in medium without IFN-β for 46 h before lysis. GAPDH immunoblotting is used to normalize protein loading. (B) Analysis of proteins associated with nascent DNA, isolated by iPOND. HEK293T cells transfected with EV or myc-ISG15 for 24 h were pulse-labeled with EdU for 10 min and then chased with thymidine for 60 min. Immunoblotting with the indicated antibodies reveals the presence of ISG15 on chromatin (H3-positive fraction) and at the replication forks (H3- and PCNA-positive fraction). (C) Representative images of ISG15 colocalization with PCNA (ISG15/PCNA), as revealed by PLA. Immunofluorescence (IF) shows protein expression and cellular distribution of ISG15 and PCNA in U2OS FIT cells (treated with 1 µg/ml doxycycline for 48 h). 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) Representative images of ISG15 colocalization with newly synthesized DNA (ISG15/EdU), labeled by the nucleotide analogue EdU (1 µM, 8 min), as revealed by PLA. Immunofluorescence (IF) shows cellular distribution of ISG15 and EdU in U2OS FIT cells (treated with 1 µg/ml doxycycline for 48 h). Scale bars, 10 µm. (F) QIBC shows the distribution of PLA foci counts of samples described in E. For each condition, images containing ≥1,000 cells per experiment were acquired (n = 3). (G) Top: DNA fibers labeling strategy and representative image. Bottom: Analysis of IdU track length measurements in U2OS FIT cells expressing EV or FLAG-ISG15 (treated with 1 µg/ml doxycycline for 48 h). At least 100 tracks were scored per sample (n = 5). 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. (H) FLAG-ISG15 expression in U2OS FIT cells after induction with 1 µg/ml doxycycline for the indicated time points. (I) Analysis of IdU track length measurements in U2OS FIT cells upon ISG15 induction as in H. At least 100 tracks were scored per sample (n = 3).$

ISG15 localizes at the DNA replication forks and accelerates replication fork progression. (A) ISG15 immunoblot on protein extracts of U2OS FIT cells bearing EV or FLAG-ISG15, induced with doxycycline (dox; 1 µg/ml) for 48 h, and in parental U2OS treated with IFN-β (30 U/ml for 2 h) and chased in medium without IFN-β for 46 h before lysis. GAPDH immunoblotting is used to normalize protein loading. (B) Analysis of proteins associated with nascent DNA, isolated by iPOND. HEK293T cells transfected with EV or myc-ISG15 for 24 h were pulse-labeled with EdU for 10 min and then chased with thymidine for 60 min. Immunoblotting with the indicated antibodies reveals the presence of ISG15 on chromatin (H3-positive fraction) and at the replication forks (H3- and PCNA-positive fraction). (C) Representative images of ISG15 colocalization with PCNA (ISG15/PCNA), as revealed by PLA. Immunofluorescence (IF) shows protein expression and cellular distribution of ISG15 and PCNA in U2OS FIT cells (treated with 1 µg/ml doxycycline for 48 h). 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) Representative images of ISG15 colocalization with newly synthesized DNA (ISG15/EdU), labeled by the nucleotide analogue EdU (1 µM, 8 min), as revealed by PLA. Immunofluorescence (IF) shows cellular distribution of ISG15 and EdU in U2OS FIT cells (treated with 1 µg/ml doxycycline for 48 h). Scale bars, 10 µm. (F) QIBC shows the distribution of PLA foci counts of samples described in E. For each condition, images containing ≥1,000 cells per experiment were acquired (n = 3). (G) Top: DNA fibers labeling strategy and representative image. Bottom: Analysis of IdU track length measurements in U2OS FIT cells expressing EV or FLAG-ISG15 (treated with 1 µg/ml doxycycline for 48 h). At least 100 tracks were scored per sample (n = 5). 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. (H) FLAG-ISG15 expression in U2OS FIT cells after induction with 1 µg/ml doxycycline for the indicated time points. (I) Analysis of IdU track length measurements in U2OS FIT cells upon ISG15 induction as in H. At least 100 tracks were scored per sample (n = 3).

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