Figure S1. ISG15 localizes at the DNA... | Rockefeller University Press

Figure S1.

$ISG15 localizes at the DNA replication forks and accelerates replication fork progression.(A) Schematic representation of the pipeline followed for the generation of CRISPR/Cas9-mediated ISG15 KO cell lines. (B) Analysis of ISG15 protein levels in 7 of the 42 single clones tested, obtained from the CRISPR/Cas9 KO in U2OS FIT cells. Clone D3 was selected and used for the following experiments (in the main text referred to as U2OS ISG15/KO). 50 µg cell extracts was analyzed by Western blotting as indicated. (C) Subcellular localization of ISG15 in U2OS FIT cells expressing EV or FLAG-ISG15 after 48 h induction with 1 µg/ml doxycycline. Indicated are the different fractions analyzed. (D) Quantification of PLA foci counts by automated microscopy (QIBC) of FLAG-ISG15 colocalization with PCNA (FLAG/PCNA) determined by PLA in U2OS FIT cells after induction with 1 µg/ml doxycycline for 48 h. For each condition, images containing ≥1,000 cells per experiment were acquired (n = 3). (E) U2OS FIT cells after doxycycline induction (1 µg/ml, 48 h) and grown in media supplemented with 10 µM EdU for 30 min before collecting and processing for FACS analysis. Left: DNA content (DAPI) and DNA synthesis, indicated by EdU incorporation (FITC) measured by FACS. Right: Quantification of EdU incorporation of cells in S phase. Similar results were obtained in at least one independent experiment. (F) Percentage of origin firing events in U2OS FIT expressing either the EV or FLAG-ISG15 after doxycycline induction (1 µg/ml, 48 h). Origin firing events were evaluated by fibers assay (IdU-CldU-IdU) scoring ≥200 DNA fibers per experiment; each point indicates a single experiment. The line connects values for EV and FLAG-ISG15 of the same experiment. (G) Graphical scheme of sister forks imaging by DNA fiber assay with representative image. (H) Sister forks symmetry plot in U2OS FIT cells after doxycycline induction (1 µg/ml, 48 h). U2OS FIT cells expressing EV were treated with CPT (50 nM, 1 h; EV + CPT) as a positive control for asymmetry. 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.$

ISG15 localizes at the DNA replication forks and accelerates replication fork progression. (A) Schematic representation of the pipeline followed for the generation of CRISPR/Cas9-mediated ISG15 KO cell lines. (B) Analysis of ISG15 protein levels in 7 of the 42 single clones tested, obtained from the CRISPR/Cas9 KO in U2OS FIT cells. Clone D3 was selected and used for the following experiments (in the main text referred to as U2OS ISG15/KO). 50 µg cell extracts was analyzed by Western blotting as indicated. (C) Subcellular localization of ISG15 in U2OS FIT cells expressing EV or FLAG-ISG15 after 48 h induction with 1 µg/ml doxycycline. Indicated are the different fractions analyzed. (D) Quantification of PLA foci counts by automated microscopy (QIBC) of FLAG-ISG15 colocalization with PCNA (FLAG/PCNA) determined by PLA in U2OS FIT cells after induction with 1 µg/ml doxycycline for 48 h. For each condition, images containing ≥1,000 cells per experiment were acquired (n = 3). (E) U2OS FIT cells after doxycycline induction (1 µg/ml, 48 h) and grown in media supplemented with 10 µM EdU for 30 min before collecting and processing for FACS analysis. Left: DNA content (DAPI) and DNA synthesis, indicated by EdU incorporation (FITC) measured by FACS. Right: Quantification of EdU incorporation of cells in S phase. Similar results were obtained in at least one independent experiment. (F) Percentage of origin firing events in U2OS FIT expressing either the EV or FLAG-ISG15 after doxycycline induction (1 µg/ml, 48 h). Origin firing events were evaluated by fibers assay (IdU-CldU-IdU) scoring ≥200 DNA fibers per experiment; each point indicates a single experiment. The line connects values for EV and FLAG-ISG15 of the same experiment. (G) Graphical scheme of sister forks imaging by DNA fiber assay with representative image. (H) Sister forks symmetry plot in U2OS FIT cells after doxycycline induction (1 µg/ml, 48 h). U2OS FIT cells expressing EV were treated with CPT (50 nM, 1 h; EV + CPT) as a positive control for asymmetry. 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.

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