Df6102

Briefly, after performing the indicated treatments, all proteins were extracted with RIPA buffer (Solarbio, Beijing, China), and protein quantification was performed with a BCA assay kit (Solarbio, Beijing, China). Protein lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). PVDF membranes were incubated overnight at 4°C with rabbit anti‐ARID1A (1:1000, 12354S, CST), rabbit anti‐CDK2 (1:10,000, ab32147, Abcam), rabbit anti‐CDK4 (1:1000, DF6102, Affinity), rabbit anti‐CDK6 (1:100,000, ab124821, Abcam), rabbit anti‐Cyclin D (1:2000, AF0931, Affinity), mouse anti‐GAPDH (1:50,000, 60004‐1‐Ig, Proteintech), rabbit anti‐BAX (1:1000, 41162S, CST), rabbit anti‐Cleaved Caspase‐3 (1:1000, 9664S, CST), rabbit anti‐Cleaved Caspase‐7 (1:1000, 8438S, CST), rabbit anti‐β‐actin (1:10,000, AF7018, Affinity), rabbit anti‐RAD50 (1:5000, 29390‐1‐AP, Proteintech), rabbit anti‐CHK2 (1:1000, 6334S, CST), rabbit anti‐RAD51 (1:2000, 14961‐1‐AP, Proteintech), rabbit anti‐γ‐H2AX (1:1000, ab229914, Abcam), rabbit anti‐BRG1 (1:1000, 49360S, CST), followed by a 1 h incubation at room temperature with the horseradish peroxidase (HRP)‐conjugated respective secondary antibody for chemiluminescence‐based protein detection.

Cells were washed with PBS and incubated with propidium iodide solution (500 μg/ml and 100 μg/ml RNaseA) for 30 min. Cells were further analyzed by flow cytometry. In addition, qPCR and WB were used to verify the effect of RAC1 (AF4200, Affinity, China) on the expression of cell cycle related genes and proteins CDK4(DF6102, Affinity, China), CDK6 (DF6448, Affinity, China), CyclinD1 (AF0931, Affinity, China).

A RIPA lysate (Beyotime, China) was utilized to extricate total proteins, which were quantified by a BCA kit (Solarbio, China), followed by subjecting them to an SDS-PAGE gel and transferring them to PVDF membranes (GE Healthcare Life, USA). Afterwards, blocking blots were performed with 5% fat-free milk, and the process was followed by incubation with the primary antibodies against KRT17 (AF5480, Affinity, USA), E-cadherin (AF0131, Affinity), N-cadherin (AF4039, Affinity), β-catenin (AF6266, Affinity), AKT (AF6261, Affinity), p-AKT (AF0016, Affinity), mTOR (AF6308, Affinity), p-mTOR (AF3308, Affinity c-Myc (AF0358, Affinity), cyclin D1 (AF0931, Affinity), cyclin E (AF0144, Affinity), CDK2 (AF6237, Affinity), CDK4 (DF6102, Affinity), Snail (AF6032, Affinity), MMP7 (AF0218, Affinity), and GAPDH (AF7021, Affinity). Then, the membranes were encoded with a secondary antibody conjugated to HRP and finally subjected to measurement with an ECL kit (Pierce, Waltham, MA, USA).