Cytoflex ow cytometer

Transfection of mESC was performed with Lipofectamine 2000 (Invitrogen) in a 24-well plate as previously described 61 . Total 2×10 5 mESC harboring the HR/NHEJ reporter were transfected with 0.5 μg total DNA. For U2OS and NIH3T3 cells, 1.0×10 5 cells were seeded on a 24-well plate and total 0.8 μg DNA were transfected by Lipofectamine 2000. To initiate DNA replication in HR reporter U2OS cells, 0.16 μg of the SV40 LT expression plasmid in 0.8 μg of total DNA was co-transfected with expression plasmids for I-SceI or CRISPR nucleases. For chemical treatment, small molecule inhibitors were added at 6 h posttransfection, and replaced with fresh ones next day for a continued treatment for the rest of the experiment. Transfected or treated cells were analyzed for GFP + RFP -, GFP + RFP + and GFP -RFP + frequencies using the Beckman Coulter CytoFLEX ow cytometer at least 3 days post-transfection. The HR/NHEJ frequencies were calculated after being corrected with background readings and normalized with transfection e ciencies as described before 60, 61 .

The cell cycle was analyzed by a kit from KeyGEN BioTECH (KGA512, Nanjing, China). Cells transfected with the PTEN vector for 48 h were harvested by digestion and centrifugation at 400 g for 5 min, and cells were further xed with 70% precooled ethanol overnight. Fixed cells were centrifuged at 400 g for 3 min and incubated with 500 µL of propidium iodide (PI)/RNase A mixture (9:1, v/v). Then, the cell cycle was detected using a CytoFLEX ow cytometer from Beckman Coulter (Brea, CA). The proliferation index was calculated as follows: Proliferation index (PI) = (S + G 2 /M) / (G 0/1 +S+G 2 /M) × 100%.

The half maximal inhibitory concentrations of the selected drugs of HAP1, HAP1 RNF43 KO and HAP1 PWWP2B KO cells were measured using the MTS assay for selected drugs at concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 µM for 48 h. On the day of the proliferation assay, medium was removed, and 100 µL of fresh medium was added to each well of 96-well plates, followed by 20 µL of MTS solution, and the plates were incubated at 37°C for 1 h in a humidi ed environment with 5% CO 2 .
The absorbance was read at 490 nm using a microplate reader (Synergy 2 Multi-Mode Microplate Readers; BioTek, Winooski, VT, USA). The IC 50 values were determined after tting growth inhibition curves to dose-response curves using GraphPad Prism software (GraphPad Software Inc., CA, USA).
Apoptosis analysis HAP1, HAP1 RNF43 KO, HAP1 PWWP2B KO, SNU620, Kato III, MKN28, and MKN45 cells seeded onto 6well plates at a density 5 × 10 4 cells per mL were treated with the respective IC50 values of docetaxel trihydrate, pelitinib, and uprosertib (Table 2). Cell death was determined using the Annexin V-APC/Propidium Iodide (PI) Apoptosis Detection Kit (Thermo Fisher Scienti c, Rockford, IL, USA) on a CytoFLEX ow cytometer (Beckman Coulter, Brea, CA, USA). The percentages of intact and apoptotic cells were calculated using CytExpert software (Beckman Coulter).

Ec109 cells (1.5 × 10 5 ) were maintained in 6-well plates at overnight and treated with ginsenoside CK or DMSO for 48 h. sh-NC and sh-VEGF-A cells (1.5 × 10 5 ) were planted in 6-well plates for 48 h. The rates of apoptosis cells were assessed by applying the Annexin V-FITC Apoptosis Detection Kit (Beyotime). The results were detected by the CytoFLEX ow cytometer (Beckman).

Cell apoptosis was analyzed by a kit from KeyGEN BioTECH (KGA108-2, Nanjing, China). After 48 h of transfection, PC3 cells in a 6-well plate were digested with trypsin without EDTA, and cells were washed with phosphate buffered saline (PBS) twice for 5 min. Next, cells were suspended in 500 µL of binding buffer and incubated with 5 µL of annexin V-FITC and 5 µL of propidium iodide for 15 min. The apoptotic rates were obtained using a CytoFLEX ow cytometer from Beckman Coulter (Brea, CA).