Fig. 3: USP50 promotes WRN-FEN1 localisation at stalled forks.

Where included, graphs indicate the mean ± SEM, exact P values are shown, and number of biological repeats is listed (n). All statistical analysis in this figure was performed using a two-tailed unpaired t test. Source data are provided with this paper. A Mean% of stalled forks after shUSP50 and WRN siRNA. n = 3, >200 fibres per condition. B Immunoblot of indicated proteins in whole-cell lysate and chromatin fraction after shUSP50 and 3 hours 5 mM HU (representative of two). C Mean proximity ligation assay (PLA) foci of endogenous WRN with HUS1 after siNTC, siWRN, siUSP50 and 3 hours 5 mM HU. n = 3, >145 cells per condition. D Mean% first-label terminations after siRNA to USP50 and GFP-WT-WRN (WT), GFP-E84A-WRN (EA) or GFP-K577M-WRN (KM) expression. n = 3, >200 fibres per condition. E Mean ratio of second-label tracts on either side of first labels after treatment as in D. n = 3, >34 first-label origins per condition. F Mean 53BP1 foci after USP50 siRNA and GFP or GFP-WRN expression. n = 3, >130 cells per condition. G Immunoprecipitation with anti-WRN or control IgG (G) after siNTC, shUSP50 and 3 hours 5 mM HU, probed for WRN or FEN1. Numbers below: ratio of FEN1:WRN relative to siNTC (performed once). H Mean PLA foci of endogenous WRN with FEN1 after siNTC, siFEN1, shUSP50, FLAG-USP50 or I141R-FLAG-USP50 expression and 3 hour 5 mM HU. n = 3, >150 cells per condition. I Immunoblot of indicated proteins input or purified by iPOND after shUSP50 and 3 hours 5 mM HU (representative of three). J Mean% stalled forks after shUSP50 and siRNA to FEN1. n = 3, >200 fibres per condition. K Mean ratio of second-label tracts, either side of first labels after shUSP50 and WT Myc-FEN1 (WT) or E359K-Myc-FEN1 (EK) expression. n = 3, >70 first-label origins measured. L Mean% of stalled forks after treatment as in K. n = 3, >200 fibres per condition. M Mean 53BP1 foci after treatment as in K. n = 3, 150 cells per condition.