Ab32103

PBS at 4 °C was used to wash the cells twice. Thereafter, protease inhibitors in cold RIPA buffer were used to lyse the cells. Protein concentration was determined using BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China). Total protein of the cells were denatured using 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used to block the membrane for 1 h at room temperature. The membranes were then incubated with the primary antibodies overnight at 4 °C (dilution ratio 1:2000). TBST was used to wash the membranes three times and they were subsequently incubated with secondary antibodies (anti-rabbit IgG or anti-mouse IgG) for 1 h at room temperature. TBST was once again used to wash the membranes three times. The targeted proteins were identified using the ECL (EMD Millipore, MA, USA) method. SKA3, CCNE2, CCNA2, CDK4, CDK2, P53, P53-pSer15, P53-pSer46 antibodies used for western blot in this research were purchased from Bioss (bs-7848R), Abcam (ab32147), Abcam (ab108357), Abcam (ab181591), Abcam (ab32103), Abcam (ab32389), CST #9284 and CST#2521.

PC9 and HCC827 cells were washed twice with ice-cold PBS and lysed with RIPA buffer (50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% Sodium deoxycholate, 0.1% SDS) (P0013B; Beyotime; China). The lysed samples were centrifuged for 15 min at a maximum speed (14000 rpm). The whole cell extracts were quantified by BCA Protein Assay kit (P0012S; Beyotime; China) and boiled for 5 min. Denatured proteins was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Millipore). Subsequently, PVDF membranes were blocked with 5% milk-powder in PBST (1×PBS containing 0.1% Tween-20) for 1 ~ 2 h and then incubated overnight with primary antibodies, including CARM1 (Abcam; ab245467), CCNE2 (Abcam; ab32103), H3R17me2a (Abcam; ab8284), H3R26me2a (Abcam; ab194679), Histone H3 (Abcam; ab1791) and GAPDH (Abcam; ab181602). After washing with PBST, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Sigma). Protein expression was visualized by enhanced chemiluminescence detection kit (Thermo Scientific Pierce). GAPDH was used as a loading control.

The RIPA lysis buffer (Beyotime, China) was used extract total proteins from clinical tissues and cell lines. The proteins were separated by 10% sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the targeted protein bands were transferred onto PVDF membrane (Sigma, USA). Subsequently, the PVDF membranes were probed with the primary antibodies including anti-β-actin (#ab8226, Abcam, USA), anti-SOD2 (#ab13534, Abcam, USA), anti-NLRP3 (#ab214185, Abcam, USA), anti-CyclinD1 (#ab16663, Abcam, USA), anti-Cyclin E2 (#ab32103, Abcam, USA), anti-CDK2 (#ab32147, Abcam, USA), anti-CDK4 (#ab108357, Abcam, USA), anti-CDK6 (#ab124821, Abcam, USA), anti-Caspase 3 (#ab13847, Abcam, USA), anti-Bax (#ab32503, Abcam, USA), anti-Bcl-2 (#ab185002, Abcam, USA), anti-Caspase 1 (#ab238979, Abcam, USA) and anti-IL-1β (#ab33774, Abcam, USA). After that, the membranes were incubated with anti-Rabbit IgG antibody at 4°C for 24h, and the enhanced chemiluminescent (ECL) system was employed to detect the protein bands. Finally, all the protein bands were quantified by Image J software. The primers sequences of the target genes were listed in Table 1.