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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Mol Cell. 2021 Feb 10;81(7):1515–1533.e5. doi: 10.1016/j.molcel.2021.01.019

Figure 3. ATM-deficient cells accumulate single-strand breaks and R-loops.

Figure 3.

(A) Human U2OS cells with ATM shRNA depletion and wild-type (WT), C2991L (CL), or R3047X (RX) ATM expression; western blotting with anti-ATM antibody and HSP90 as loading control. Lower panel: U2OS cells with ATM shRNA depletion and inducible expression of V5-tagged Senataxin C-terminus and β-actin as loading control. (B) Alkaline comet assays were performed in U2OS cells with ATM depletion, expressing WT, CL, or RX ATM alleles with NAC treatment (1 mM) as indicated. Examples of comets are shown. (C) Quantitation of the olive moment in > 200 cells from each treatment group; error bars represent SEM. (D) Quantification of ROS levels in U2OS cells with endogenous ATM depletion and induced expression of WT, CL, or RX alleles of ATM; measured by CellROX in triplicate. (E) Alkaline comet assays as in (B,C) with 1 day DRB treatment (20 μM). (F) Alkaline comet assays as in (B,C) with ATM shRNA depletion and inducible expression of SETX. (G) DRIP-qPCR assays performed in triplicate with primers specific for the BTBD19, β-actin, or EGR1 loci in U2OS cells depleted for endogenous ATM and expressing WT, CL, or RX alleles as indicated. Immunoprecipitations were performed without S9.6 antibody or with RNaseH treatment in vitro to verify assay specificity. Levels of product were normalized to the level obtained in WT-expressing cells. Error bars indicate standard deviation. (H) DRIP-qPCR assays performed in triplicate as in (G) with addition of 1 mM NAC. (I) DRIP-qPCR assays performed in triplicate as in (G) with SETX expression indicated. *, **, ***, and **** indicate p<0.05, 0.005, and 0.0005 by Student two-tailed t-test; NS = not significant.