Figure 5.
High levels of ISG15 unleash DNA replication, induce DNA breakages and sensitize cells to genotoxic stress.(A) Top: Schematic representation of DNA fiber assay strategy. Bottom: Effect of CPT treatment (50 nM), optionally added concomitantly with the second label (IdU), in U2OS FIT cells expressing EV, FLAG-ISG15 or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h). Drug effect (IdU + CPT) is normalized on CldU track length in untreated conditions. At least 100 tracks were scored per sample (n = 3). Horizontal 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. (B) Analysis of IdU track length measurements in cells treated as in (A). 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. (C) Effect of CPT treatment (50 nM) in U2OS FIT cells expressing EV or FLAG-ISG15 after dox-induction (1 µg/ml, 48 h) and upon RECQ1 knockdown (siRECQ1). Drug effect (IdU + CPT) is normalized on CldU track length in untreated conditions. At least 100 tracks were scored per sample (n = 3). Horizontal 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. (D) Neutral comet assay to measure accumulation of DNA double strand breaks in U2OS FIT cells expressing EV, FLAG-ISG15 or FLAG-ISG15ΔGG induced with doxycycline (1 µg/ml) for 48 h and treated with CPT (50 nM; n = 3). (E) Quantification of chromosomal abnormalities by analysis of metaphase spreads of U2OS FIT cells expressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h) and treated with CPT (50 nM). At least 50 metaphases/sample were scored for every replicate. Each spot represents average of one replicate (n = 3). (F) Representative image of chromosomal abnormalities quantified in (E). Scale bar of 10 µm is indicated. (Gand H) Survival curve determined by clonogenic assay of U2OS FIT cells expressing EV or FLAG-ISG15 and treated with increasing doses of CPT (G) and the PARP inhibitor Olaparib (H). Values are normalized on untreated and EV (n = 3).

High levels of ISG15 unleash DNA replication, induce DNA breakages and sensitize cells to genotoxic stress. (A) Top: Schematic representation of DNA fiber assay strategy. Bottom: Effect of CPT treatment (50 nM), optionally added concomitantly with the second label (IdU), in U2OS FIT cells expressing EV, FLAG-ISG15 or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h). Drug effect (IdU + CPT) is normalized on CldU track length in untreated conditions. At least 100 tracks were scored per sample (n = 3). Horizontal 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. (B) Analysis of IdU track length measurements in cells treated as in (A). 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. (C) Effect of CPT treatment (50 nM) in U2OS FIT cells expressing EV or FLAG-ISG15 after dox-induction (1 µg/ml, 48 h) and upon RECQ1 knockdown (siRECQ1). Drug effect (IdU + CPT) is normalized on CldU track length in untreated conditions. At least 100 tracks were scored per sample (n = 3). Horizontal 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. (D) Neutral comet assay to measure accumulation of DNA double strand breaks in U2OS FIT cells expressing EV, FLAG-ISG15 or FLAG-ISG15ΔGG induced with doxycycline (1 µg/ml) for 48 h and treated with CPT (50 nM; n = 3). (E) Quantification of chromosomal abnormalities by analysis of metaphase spreads of U2OS FIT cells expressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after doxycycline induction (1 µg/ml, 48 h) and treated with CPT (50 nM). At least 50 metaphases/sample were scored for every replicate. Each spot represents average of one replicate (n = 3). (F) Representative image of chromosomal abnormalities quantified in (E). Scale bar of 10 µm is indicated. (Gand H) Survival curve determined by clonogenic assay of U2OS FIT cells expressing EV or FLAG-ISG15 and treated with increasing doses of CPT (G) and the PARP inhibitor Olaparib (H). Values are normalized on untreated and EV (n = 3).

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