Nb100 464

The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10565-018), fetal bovine serum (Gibco, 10082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), phosphatase inhibitor cocktail (Sigma, P0044), protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCHEK1 lentivirus particles (pGFP-C-shLenti, Origene, TR320302), CHIR-124 (Selleckchem, S2683), anti-CHEK1 (Novus Biologicals, NB100-464, rabbit, used for WB and IHC), anti-RAD54L (Novus Biologicals, NBP2-33916, rabbit, used for WB), and β-Actin (Sigma, A5316, mouse, used for WB).

Adherent cells in the tissue culture wells were rinsed with PBS and directly lysed in the RIPA buffer. 50 μg of cell lysate was fractionated on 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF membrane). The PVDF membrane was blocked with 5% nonfat dry milk and probed with specific antibodies against hRad9 (1:5000, NBP1-47946, Novus Biologicals, LLC, USA) and chk1 (1:5000, NB100-464, Novus Biologicals, LLC, USA). Expression of GAPDH protein was assessed as an internal control by using anti-GAPDH antibody (1:200, TA-08, ZSGB-BIO, Beijing, China). Enhanced chemiluminescent detection (ECL) was performed according to the vendor's protocols (Santa Cruz Biotechnology).

All the samples were fixed with 10% (volume/volume) neutral-buffered formalin overnight and then embedded in paraffin. Sections were deparaffinized and subjected to antigen retrieval by autoclave in 0.01 m EDTA buffer (pH 8.5) for 5 min, followed by incubation in 3% H2O2 for 10 min to quench endogenous peroxidase. Primary antibodies for Immunohistochemistry (IHC) included specific antibodies against hRad9 (NBP1-47946, Novus Biologicals, LLC, USA) with a dilution of 1:1000, chk1 (NB100-464, Novus Biologicals, LLC, USA) with dilution of 1:300 and cyclinD1 (ZA-0101, ZSGB-BIO, Beijing, China) with a dilutin of 1:30. Sections were incubated overnight at 4°C with primary antibodies diluted in 1% BSA. Substitution of the primary antibody with PPS served as a negative control. After washed with PBS for 3 times and incubated with secondary antibody (EnVision™ Detection Systems K5007, Dako Denmark A/S) at room temperature for 0.5 h, staining was carried out by incubating the slides in 3, 3′-diaminobenzidine (DAB), followed by counterstained with hematoxylin, dehydrated in alcohol and xylene. Immunohistochemistry for other biomarkers, such as ER, PR, HER2 and Ki67 was done and evaluated in the department of Pathology at Shandong University by Roche Benchmark XT automated slide preparation system (Roche Ltd, Switzerland) according to the ultraview DAB v3 procedure.