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. 2020 Nov 13;6(46):eabc3598. doi: 10.1126/sciadv.abc3598

Fig. 4. FBH1 helicase activity is needed to generate the fork protection substrate for 53BP1.

Fig. 4

(A to C) Fork protection assays and analyses were completed as in Fig. 1. (A) WT U2OS cells were transfected with the indicated siRNAs. (B) WT U2OS cells were transfected with the indicated siRNAs and transfected with expression vectors encoding WT, F-box mutant (FM), or helicase mutant (HM) FBH1 before performing the fork protection assay. Immunoblots for 53BP1 and GFP-FBH1 are shown. (C) FBH1 KO U2OS cells were complemented with empty vector (EV), WT, FM, or HM FBH1-expressing retroviruses before depleting 53BP1 with siRNA and analyzing fork protection. HA-FBH1 protein expression was examined by immunoblotting. (D) Quantitation of the γH2AX nuclear intensity in EdU-positive cells transfected with the indicated siRNAs. Bars represent the mean, and P values were derived from a Kruskal-Wallis test. (E) U2OS cells transfected with the indicated siRNAs were treated with HU for 48 hours after plating for individual colonies. Colony number was scored after 10 days (mean ± SD, n = 3).