Fig. 5: NSC617145 directly binds WRN and causes WRN trapping on DNA.

a Dot blots showing in vitro binding of radiolabeled ¹⁴C-NSC617145 (10 µM) to recombinant WRN or RECQL1 proteins (300 nM). Quantitation of the data obtained from the binding experiments. Data represent mean ± SEM of four repeats. One-way ANOVA with Bonferroni’s multiple comparison test: p = 0.0003, p = 0.0058, p = 0.5744. b ¹⁴C-NSC617145 binds to WRN proteins in U2OS cells. Immunoblots showing WRN protein levels in whole-cell extract and α-FLAG immunoprecipitates prepared from U2OS/WRN^−/− cells transfected with either empty FLAG vector or FLAG-WRN expression plasmids. IgG heavy chains (HC) are shown. α-FLAG immunoprecipitates prepared from ¹⁴C-NSC617145 (10 μM) treated cells were subjected to dot blot assay. The bar graph represents ¹⁴C-NSC617145 signal intensity. Data represent the mean of two independent experiments. c Immunoblots showing WRN chromatin enrichment in PEO1 cells treated with indicated doses of NSC617145 for 3 h. ORC2 was used as a positive marker and loading control for chromatin fractions. The experiment was repeated three times with similar outcomes. d Relative chromatin enrichment of WRN in PEO4 and PEO1 cells upon WRNi treatment for 1 h. Bar graph showing WRN levels in chromatin-bound fractions prepared from PEO4 and PEO1 cells. WRN signal intensity was normalized to ORC2 and represented as relative fold change over PEO4 untreated cells (set as one). Data represent the mean of two independent experiments. e Immunoblots showing enhanced chromatin enrichment of WRN upon NSC617145 exposure in presence of HU. PEO1 cells were treated with indicated doses of NSC617145 with or without HU (2 mM) for 2 h and subjected to sub-cellular fractionation. Bar graph represents quantification of WRN chromatin enrichment. Data represent the mean of two independent experiments. f Persistent trapping of WRN on chromatin by WRNi. PEO1 cells were treated with NSC617145 (6 μM) for 2 h before removal of the drug by ice-cold PBS wash. Cells were incubated further in a pre-warmed cell culture medium for the indicated times. Soluble and chromatin fractions were probed with the indicated antibodies. The experiment was repeated independently two times with similar results. Source data are provided as a Source Data file.