Figure 4.

Loss of SMARCA4 increases intrinsic DNA replication stress and ATRi susceptibility. (A–E) SMARCA4^WT cells and SAECs were transfected with control siRNA (siCTL) or two independent SMARCA4 siRNAs. Forty-eight hours later, whole-cell lysates (WCLs) were prepared and the expression of SMARCA4 was analyzed by western blotting (A). Analysis of the velocities (B) and asymmetries (C) of individual forks by DNA fiber assays. Left- and right-moving sister fork (CldU tracks) lengths were measured and plotted. The central areas marked by red lines indicate sister forks with differences in length <30%. The percentages of each outlier, defined as asymmetric forks, are shown in each panel at bottom right (C). Representative results of two independent reproducible experiments are shown. (D) Quantification of the intensities of exposed ssDNA in 200 S-phase PC9 cells and SAECs transfected with siCTL or siSAMRCA4. Red bars represent the mean intensities. Representative results of two independent reproducible experiments. (E) Forty-eight hours after transfection with siCTL and siSMARCA4, the cells were re-seeded, incubated for 24 h and treated with varying doses of ATRi. Cell viability was measured using the PrestoBlue assay. The results represent the mean ± SD of three independent experiments. (F–J) SMARCA4^MUT cells were lentivirally transduced with SMARCA4 or empty vector (EV). WCLs were prepared and SMARCA4 levels were analyzed by western blotting (F). Analysis of the velocities (G) and asymmetry (H) of individual DNA fibers from A549 cells transduced with EV or SMARCA4, as described in (B) and (C). (I) Quantification of the ssDNA intensities of 200 S-phase A549 cells transduced with EV or SMARCA4 using the protocol described in (D). (J) A549 cells transduced with EV or SMARCA4 were treated with various concentrations of ATRi, and cell viability was measured by the PrestoBlue assay. The results represent the mean ± SD of three independent experiments.