Cyclin a1

A RIPA (radioimmunoprecipitation) lysis buffer from Beyotime (Shanghai, China) was used to isolate total proteins from cultured RA-FLSs. The determination of the concentration of total protein was carried out employing a BCA protein assay kit procured from Pierce (Rockford, IL, U.S.A.). Proteins in equal, and measured amounts were electrophoretically separated on SDS-PAGE (10%) and transferred to PVDF membranes (polyvinylidene fluoride; Millipore, U.S.A.). After the blocking procedure with nonfat milk (5%), antibodies against TLR4 (1:2000), CyclinA1(1:1000), CyclinB1(1:1000), CyclinD2(1:2000), and GAPDH (1:5000) (all from Santa Cruz, U.S.A.) were added to the membrane, followed by secondary antibodies (1:6000, Santa Cruz) conjugated with horseradish peroxidase. The internal loading control was GAPDH. The chemiluminescent substrate kit from Millipore Company (Bedford, MA, U.S.A.) was used to observe protein bands as per instructions of the manufacturer. Gray analysis was performed using software Gel-Pro Analyzer 4 (United States Biochemical, Cleveland, OH, U.S.A.).

PD-0332991 (palbociclib), a selective CDK4/6 inhibitor, was purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-phospho-H2AX (Ser139) antibodies were purchased from Millipore (Billerica, MA, USA). RB1, phospho-RB1 (Ser780; Ser807/811), E2F1, cyclin B1, cyclin D1, cyclin E1, CDK4, and CDK6 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibodies were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-RAD51, cyclin A1, and CDKN2A antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Total protein or nuclear/cytoplasmic protein fractions were extracted from cells using lysis buffer (Beyotime Biotechnology Shanghai, China) supplemented with protease inhibitor. Western blot was performed with a standard method as previously described (Chen et al., 2018b) using the following antibodies: RDM1 (1 : 500; Proteintech, Rosemont, IL, USA), p53 (1 : 2000; Santa Cruz Biotechnology, Dallas, TX, USA, β‐actin (1 : 2000; Santa Cruz), 14‐3‐3σ (1 : 1000; Santa Cruz), p21 (1 : 2000; CST, Boston, MA, USA), Cyclin A1 (1 : 1000; Santa Cruz), Cyclin B1 (1 : 1000; CST), VEGFB (1 : 1000; CST), BAX (1 : 1000; CST), RAD51 (1 : 1000; CST), hTERT (1 : 500; Abcam, UK), c‐myc (1 : 1000; Proteintech), METTL3 (1 : 1000; Abcam), METTL14 (1 : 1000; Abcam), Ras (1 : 500; CST), p‐cRaf (1 : 500; CST), ERK (1 : 1000; CST), and p‐ERK (1 : 1000; CST).