Figure 2.
ISG15 expression levels impact on replication fork progression in different systems.(A) Time course of ISG15 expression in U2OS treated with IFN-β (30 U/ml, 2 h) and chased for the indicated time points before lysis. (B) Analysis of IdU track length measurements in U2OS cells treated with IFN-β as in A. At least 100 tracks were scored per sample (n = 3). (C) ISG15 protein levels in U2OS FIT cells carrying ISG15 WT or ISG15 KO treated with IFN-β (30 U/ml, 2 h) and chased for 46 h before lysis. Phosphorylated STAT1 (pSTAT1) reveals activation of IFN-β pathway. Immunoblot with STAT1 and GAPDH are used to normalize protein loading. (D) Top: DNA fiber–labeling strategy. Bottom: Analysis of IdU track length measurements in U2OS as in C. 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. (E) ISG15 immunoblot of parental U2OS FIT cells expressing EV or FLAG-ISG15 and U2OS FIT cells lacking the endogenous ISG15 (U2OS ISG15/KO) and reexpressing stably integrated EV or exogenous FLAG-ISG15 after 48 h induction with 1 µg/ml doxycycline. Western blot analysis reveals the expression of endogenous (black triangle) and exogenous (white triangle) ISG15. (F) Analysis of IdU track length measurements in U2OS 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; ****, P < 0.0001. (G) Top: ISG15 expression in MCF7 cells carrying ISG15 WT or ISG15 KO treated with IFN-β (30 U/ml, 2 h) and chased for 46 h before lysis. Bottom: Analysis of IdU track length measurements. 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; ****, P < 0.0001; *, P < 0.05. (H) Top: ISG15 protein levels in MCF7 cells bearing the WT gene of ISG15 (MCF7) and cells lacking the endogenous ISG15 (MCF7 ISG15/KO) stably integrated with EV or FLAG-ISG15. Bottom: Analysis of IdU track length measurements. 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; ****, P < 0.0001; *, P < 0.05. (I)ISG15 knockdown (siISG15) 48 h after siRNA transfection in HeLa, T98G, and M059K cells. siLuc is used as control. (J) Analysis of IdU track length measurements as in I. 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; ****, P < 0.0001.

ISG15 expression levels impact on replication fork progression in different systems. (A) Time course of ISG15 expression in U2OS treated with IFN-β (30 U/ml, 2 h) and chased for the indicated time points before lysis. (B) Analysis of IdU track length measurements in U2OS cells treated with IFN-β as in A. At least 100 tracks were scored per sample (n = 3). (C) ISG15 protein levels in U2OS FIT cells carrying ISG15 WT or ISG15 KO treated with IFN-β (30 U/ml, 2 h) and chased for 46 h before lysis. Phosphorylated STAT1 (pSTAT1) reveals activation of IFN-β pathway. Immunoblot with STAT1 and GAPDH are used to normalize protein loading. (D) Top: DNA fiber–labeling strategy. Bottom: Analysis of IdU track length measurements in U2OS as in C. 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. (E) ISG15 immunoblot of parental U2OS FIT cells expressing EV or FLAG-ISG15 and U2OS FIT cells lacking the endogenous ISG15 (U2OS ISG15/KO) and reexpressing stably integrated EV or exogenous FLAG-ISG15 after 48 h induction with 1 µg/ml doxycycline. Western blot analysis reveals the expression of endogenous (black triangle) and exogenous (white triangle) ISG15. (F) Analysis of IdU track length measurements in U2OS 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; ****, P < 0.0001. (G) Top: ISG15 expression in MCF7 cells carrying ISG15 WT or ISG15 KO treated with IFN-β (30 U/ml, 2 h) and chased for 46 h before lysis. Bottom: Analysis of IdU track length measurements. 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; ****, P < 0.0001; *, P < 0.05. (H) Top: ISG15 protein levels in MCF7 cells bearing the WT gene of ISG15 (MCF7) and cells lacking the endogenous ISG15 (MCF7 ISG15/KO) stably integrated with EV or FLAG-ISG15. Bottom: Analysis of IdU track length measurements. 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; ****, P < 0.0001; *, P < 0.05. (I)ISG15 knockdown (siISG15) 48 h after siRNA transfection in HeLa, T98G, and M059K cells. siLuc is used as control. (J) Analysis of IdU track length measurements as in I. 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; ****, P < 0.0001.

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