SETX-deficient cells show higher R-loops and slower growth which are rescued by human RNaseH1 overexpression. (A) Laser micro-irradiation was performed in live U2OS cells stably expressing bacterial RNaseH (D20R-E48R)-mCherry as R-loop sensor. The circle indicates the site of laser damage. (B) RNaseH (D20R-E48R)-mCherry signal at laser damage sites at different time points was quantified from >8 control or SETX-depleted cells as in A. (C) Proliferation of U2OS cells with control or SETX shRNA treatment and induction of wild-type SETX, monitored by MTT assay. (D) Proliferation of U2OS cells with control or SETX shRNA treatment and induction of human RNaseH1, monitored by MTT assay as in C. (E) Slot blot analysis of genomic DNA isolated from U2OS cells with SETX knock-out (KO) and wild-type human RNaseH1 overexpression as indicated, using S9.6 and dsDNA antibodies to measure RNA-DNA hybrids and dsDNA, respectively. Recombinant RNaseH treatment confirms the specificity of the S9.6 antibody. (F) Levels of RNA-DNA hybrid signal (S9.6) were quantified in three replicates of E, normalized by dsDNA signal; shown relative to control cells. Error bars indicate standard deviation. (B–D) Error bars indicate standard error. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively, by two-tailed t test assuming unequal variance; ns = not significant. Source data are available for this figure: SourceData F4.