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: CtIP protein level is reduced in Aquarius-depleted cells. (a) Downregulation of CtIP protein level in AQR-knockdown cells. siRNAs against AQR were transfected into five different cell lines, and the cells were then lysed and CtIP and Aquarius were analysed by western blotting 48 h after siRNA transfection. (b) Partial rescue of CtIP by AQR add-back. Aquarius-depleted HCT116 cells transiently expressed GFP (EV) or GFP-tagged Aquarius (WT or HD), and CtIP protein was analysed by western blotting. (c) Analysis of CtIP mRNA by RT-PCR. Total RNA was isolated from HCT116 cells transfected with control siRNA or AQR siRNA, and PCR was performed with appropriate sets of primers after reverse transcription. RT-PCR against ß-actin (ACTB) was performed as a loading control. (d) Protein stability of CtIP in AQR-knockdown cells. U2OS and HCT116 cells were treated with CHX (20 µg/ml) and MG-132 (MG, 10 µM) for the indicated periods 48 h after control siRNA or AQR siRNA transfection. Aquarius and CtIP protein levels were analysed by western blotting. Protein levels of CtIP were normalized by ß-actin and the relative protein levels of CtIP are shown as a line graph. Data represent mean ± SD from at least three independent experiments. (e and f) Percentage of 53BP1 foci (>5)- and Rad51 foci (>10)-positive cells in AQR-knockdown HeLa (e) and U2OS (f) cells in response to MMC (200 ng/ml, 4 h) or NCS (20 ng/ml, 4 h). (g) Percentage of Rad51 foci (>10)-positive cells in CtIP overproduced AQR-knockdown cells. HCT116 cells, depleted for Aquarius, transiently expressed GFP (EV) or GFP-tagged CtIP (GFP-CtIP), and were immunostained with anti-Rad51 antibody after NCS (20 ng/ml, 4 h) treatment. Data represent mean ± SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.005.

Fig 4: SIAH2 interacts with CtIP. (A) HEK293T cells were transfected with Flag-SIAH2 along with or without HA-CtIP. Total cell lysates were immunoprecipitated with either control IgG or anti-Flag antibody and then detected with the indicated antibodies. (B) HeLa cells were lysed either 30 min after ionizing radiation (IR) or 3 h after hydroxyurea (HU) treatment and subjected to immunoprecipitation followed by immunoblotting with the indicated antibodies. (C) Interaction between Flag-SIAH2 and endogenous CtIP in either G1 or S/G2 phase of the cell cycle. Hela cells transfected with Flag-mock or Flag-SIAH2 were synchronized in G1 and S/G2, cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotting was performed with the indicated antibodies. (D) Schematic representation of the mutant CtIP constructs used in this study (E) HEK293T cells were cotransfected with each of the indicated HA-CtIP constructs and Flag-SIAH2 and subjected to immunoprecipitation and immunoblotting with the indicated antibodies. Asterisks indicate nonspecific bands. (F) Schematic representation of SIAH2 constructs used in this study. (G) Cellular lysates from HEK293T cells cotransfected with the indicated Flag-SIAH2 constructs and HA-CtIP, immunoprecipitated with anti-HA antibody and then immunoblotted with indicated antibodies. Asterisks indicate nonspecific bands. (H) Purified GST-SIAH2 WT, GST-SIAH2 ΔSBD, and GST-SIAH2 SBD expressed in bacteria were incubated with lysates from HeLa cells, and CtIP that coprecipitated with each GST-SIAH2 construct was analyzed by immunoblotting using the CtIP antibody. GST immunoblots show input GST-SIAH2 constructs. Asterisks indicate GST-SIAH2. (I) Purified GST-CtIP (700–799) expressed in bacteria was incubated with lysates from HeLa cells, and SIAH2 that coprecipitated with GST-CtIP (700–799) fragment was analyzed by immunoblotting using the SIAH2 and GST antibodies. GST immunoblots show input GST-CtIP constructs.

Fig 5: SIAH2 stimulates CtIP recruitment to DSB sites. (A) HeLa cells were either untreated or treated with 5 Gy of IR, fixed at 1 h, and immunostained with anti-SIAH2 and anti-?-H2AX antibodies. Colocalization of SIAH2 (red) and ?-H2AX (green) in cells appears yellow in the merged image. DNA was stained with DAPI. The percent colocalization between SIAH2 and ?-H2AX is shown. Results are shown as the mean ± SD (n = 3), **P < 0.01, two-tailed Student's t-test. (B) Control and SIAH2-depleted HeLa cells were either untreated or treated with 5 Gy IR. At the indicated time point after IR, chromatin and soluble fractions were extracted and analyzed by western blotting. (C) Control and SIAH2-depleted HeLa cells were either untreated or treated with 5 Gy IR, fixed at 1 h, and immunostained with anti-CtIP and anti-SIAH2 antibodies. DNA was stained with DAPI. Representative images and the percentage of cells containing >10 CtIP foci are shown. The numbers were relative levels of SIAH2 expression compared to untreated control siRNA-transfected cells. Data represent mean ± SD (n = 3), **P < 0.01, two-tailed Student's t-test. (D) The number of CtIP foci was measured 1 h after IR treatment in control Hela cells or SIAH2-depleted HeLa cells reconstituted with Flag-Mock, Flag-SIAH2 WT, or Flag-SIAH2 ?SBD. Representative images and the percentage of cells containing >10 CtIP foci in Flag-positive cells are shown. Asterisks indicate nonspecific bands.