γh2ax

OV90 and U2OS cells growing on coverslips were transfected with scrambled (siCTL) or KRCC1 siRNA (siKRCC1). For replication stress evaluation, the cells were fixed followed by immunofluorescence of RPA2 foci formation (Bethyl Laboratories, 1:500). For RAD51 foci formation evaluation, the cells were treated with camptothecin (CPT, 1 µM) for 1 h, washed with fresh warm media and collected after 2 h for the immunofluorescence of RAD51 foci formation (Novus, 1:200). Results were quantified as the percentage of the cells with >10 foci in each treatment. Statistical analysis was performed using Student’s t test (n = 3). Differences were considered significant at P < 0.05.
For RAD51 foci dynamics, OV90 cells were allowed to recover for up to 8 or 24 h and collected at indicated time points for the immunofluorescence of γH2AX (Cell Signaling Technology, 1:300) and RAD51 foci formation, which was quantified as the percentage of the cells with >10 γH2AX and RAD51 foci in each treatment. Statistical analysis was performed using two-way ANOVA (n = 3). Differences were considered significant at P < 0.05.

Cells were fixed with 4% formaldehyde in PBS after drug treatment, blocked using 5% BSA, and permeabilized with 0.2% Triton X‐100. The primary antibodies were diluted in 1% BSA and incubated at 4°C overnight. Then, secondary antibodies were added to the samples and incubated at room temperature for 1 hour. Antibodies against RAD51 (Proteintech, 14961‐1‐AP, 1:200) and γH2AX (Cell Signaling Technology, #2577,1:200) were used as the primary antibodies. Antifade Mounting Medium with DAPI was from Beyotime (China). Fluorescent secondary antibodies were used and images were captured with a fluorescence microscope (Olympus, Japan).

Cells were plated on coverslips for 24 h prior to transfection with siRNAs or DNA plasmids. After experimental treatment, cells were fixed for 30 min in 4% PFA, washed with PBS three times, and permeabilized and blocked with 0.25% Triton X-100 and 10% BSA in PBS for 1 h. Cells were then incubated with primary antibody overnight at 4 °C and with fluorescence-conjugated secondary antibody for 1 h at room temperature. After DAPI (Thermo Fisher Scientific, Waltham, MA) staining for 5 min, cells were mounted onto glass slides. Staining was assessed using a Zeiss LSM 710 immunofluorescence microscope and ZEN software (Carl Zeiss, Oberkochen, Germany). Cells with ≥5 foci per cell were classified as foci-positive. A minimum of 300 cells were counted for each experimental repeat. Representative images were obtained at ×100 magnification. The fluorescence intensity of pictures was analyzed by Image Studio version 5.0. Antibodies used include γH2AX (Cell Signaling Technology, #2577, 1:2000), 53BP1 (Bethyl Laboratories, A300-272, 1:2000), and PRMT5 (Millipore, 07–405, 1:1000).

The design and synthesis of HJC0152 were described in our previous publication.19 Antibodies against STAT3 (Cat# 4904), STAT1 (Cat# 9172), STAT5 (Cat# 9363), γ‐H2AX (Cat# 9718) and Alexa Fluor 555 goat anti‐rabbit immunoglobulin G (H + L) (Cat# 4413) were procured from Cell Signaling Technology. Bcl2 (Cat# 12789‐1‐AP) and GAPDH (Cat# HRP‐60004) antibodies were obtained from Proteintech. An antibody against STAT3 phosphorylated at tyrosine residue 705 (p‐STAT3(Tyr705)) (Cat# ab76315) was purchased from Abcam. ProLong Gold antifade reagent with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Cat# P36941) was obtained from Thermo Fisher Scientific. All other reagents used were purchased from commercial sources unless otherwise indicated. All reagents were dissolved and used as recommended by their suppliers.

Western blot analysis was carried out as previously described [12 (link), 31 (link), 32 (link)]. Primary antibodies used are as follows: HSP90, Gapdh (Ambion), γH2AX, GATA3, CtIP (Cell Signaling). For qRT-PCR, total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol and cDNA was generated using the RT Kit (YEASEN, Shanghai, China). qRT-PCR was performed as previously reported [35 (link)].