Chemiluminescence kit

Total proteins were extracted by RIPA buffer supplemented with PMSF (Solarbio, Beijing, China) and quantified by a BCA kit. Then, the proteins were separated in SDS-PAGE gels and transferred into PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with TBST with 5% skim milk powder and incubated overnight at 4 °C with primary antibodies against YAP1 (1:1000), P-YAP1 (1:1000), VEGFA (1:1000), Hexokinase II (HK2; 1:5000), LDHA (1:2000), HIF1A (1:1000), GAPDH (1:5000), Tublin (1:1000), and β-actin (1:1000) (Proteintech, Wuhan, China). The next day, blots were washed with PBS and then secondary antibodies were incubated with the membrane at room temperature for 1 h. The membrane was visualized using a chemiluminescence kit (Absin, Shanghai, China) and quantified by densitometry analysis using ImageJ software. GAPDH and tubulin were used as loading controls.

Key antibodies used included anti-S100A4 (16105-1-AP, Proteintech, Wuhan, China); β-actin (20536-1-AP, Proteintech, Wuhan, China); anti-GSDMD-N (39754S, Cell Signaling Technology, Danvers, MA, USA); anti-Caspase1 p48 (3866S, Cell Signaling Technology, Danvers, MA, USA); anti-ASC (13833S, Cell Signaling Technology, Danvers, MA, USA); anti-NLRP3 (15101S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-1β p17 (83186S, Cell Signaling Technology, Danvers, MA, USA); anti-IL-18 (54943S, Cell Signaling Technology, Danvers, MA, USA).
A total protein extraction kit was used to extract protein from cells. The protein levels were then quantified and separated using SDS-PAGE before being transferred onto an appropriate membrane (300 mA, 2 h). The blots were blocked for 1 h at room temperature, followed by overnight probing with antibodies specific for β-actin (1:3000), S100A4 (1:1000), GSDMD (1:1000), NLRP3 (1:1000), Caspase-1 p48 (1:1000), ASC (1:500), IL-1β (1:1000), IL-18 (1:1000), NF-κB/p-NF-κB (1:1000) at 4 °C. Blots were probed with an HRP-conjugated secondary antibody (1:3000) for 1 h at room temperature. Protein bands were detected using a chemiluminescence kit (Absin, Shanghai, China) and an Amersham Imager 6000.

Total protein was extracted with RIPA buffer containing PMSF (Solarbio, Beijing, China) and quantified with a BCA kit. Then, the proteins were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Massachusetts, USA). The PVDF membranes were blocked with TBST containing 5% skim milk powder and incubated with anti-YAP (No. 14074), anti-phospho-YAP (Ser127) (No. 13008), and anti-SMAD3 (No. 9523) antibodies from Cell Signaling Technology (MA, USA) and anti-ZO-1 (No. 21773-1-AP), anti-ZEB1 (No. 21544-1-AP), and anti-E-cadherin (No. 20874-1-AP) antibodies from Proteintech (Wuhan, China) at 4 °C overnight. Secondary antibodies were hybridized with the membranes at room temperature for 1 h. A chemiluminescence kit (Absin, Shanghai, China) was used to visualize the membrane.