Fig 2: ATRi-induced ssDNA depends on replication fork reversal and Mre11 activity in SMARCA4-deficient cells. (A) SMARCA4MUT (A549 and H1299) and SMARCA4WT (LC-OK) cells were pretreated with the Mre11 inhibitor Mirin (50 μM) for 1 h, followed by treatment with ATRi (1 μM) for 2 h, and the exposed ssDNA intensities of 200 S-phase cells were quantified. Red bars represent the mean intensities. The results shown are representative of two independent reproducible experiments. (B) Analysis of exposed ssDNA in SMARCA4WT PC9 cells transfected with siCTL or two independent SMARCA4 siRNAs for 48 h, pretreated or not with Mirin (1 h, 50 μM), followed by ATRi treatment (2 h, 1 μM). Quantification of the intensities of exposed ssDNA in 200 S-phase cells. Red bars represent mean intensities. The results shown are representative of two independent reproducible experiments. (C) SMARCA4MUT A549 and SMARCA4WT LC-OK cells were transfected with two independent SMARCAL1 or CtIP siRNAs for 48 h, and the levels of SMARCAL1 and CtIP were analyzed by western blotting. (D) Quantification of the intensities of exposed ssDNA in 200 S-phase A549 cells transfected with the indicated siRNAs and treated with ATRi and/or Mre11 inhibitor as in (A). Red bars represent the mean intensity. The results shown are representative of two independent reproducible experiments. (E) Quantification of the intensities of exposed ssDNA in 200 S-phase LC-OK cells transfected with the indicated siRNAs and treated with ATRi. Red bars represent the mean intensity. The results shown are representative of two independent reproducible experiments. (F, G) A549 cells transduced with EV or SMARCA4 were transfected with the indicated siRNAs. After 48 h, the cells were incubated with 10 μM EdU for 15 min and treated with 1 μM ATRi for 2 h. (F) Quantification of the intensities of total Mre11 in 200 S-phase cells. Red bars represent the mean intensities. The results shown are representative of two independent reproducible experiments. (G) Representative images of Mre11 and EdU staining of indicated cells. The nuclei were counterstained with DAPI. Scale bar: 10 μm.

Fig 3: Inhibition of fork reversal rescues fork degradation in GRB2 deficient cells.a Overexpression of BRC4 has no further fork degradation effect on GRB2 deficient cells. b BRCA2 knockdown has no further fork degradation effect on GRB2 deficient cells. c Knockdown of HTLF, SMARCAL1 or ZRANB3 is sufficient to alleviate replication fork degradation in GRB2-depleted cells. d Knockdown of RAD51 rescues the observed fork degradation in GRB2-depleted cells. e Overexpression of K133R ATPase deficient mutant RAD51 is sufficient to prevent fork degradation in GRB2-depleted HAP1 cells. f Western blot analysis showing the expression of K133RRAD51 mutant in WT and GRB2 KO HAP1 cells using anti-Flag antibody. Antibody targeting GRB2 was used to distinguish WT from GRB2 KO cells. Beta-Actin serves as a loading control. In (a–e), radiometric analysis of ≥200 fibers, n = 3. The significance was analyzed by two-sided Student’s t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; NS, not significant. Error bars showing standard deviations (SD). For (a–f), source data are provided as a Source Data file.