Pro il 18

The processed OSCC cells and the ground tumors were increased with the lysates including RIPA (Beyotime, China) and protease inhibitors (Beyotime, China). The extracted proteins were quantified through BCA method, mixed with appropriate loading, and heated at 100℃ for denaturation. Then same amount (50 μg) of proteins were added to 10% SDS-PAGE, separated by electrophoresis at constant pressure, and transferred to PVDF membrane (Millipore). Next, the membrane with protein was sealed with 5% skim milk for 2 h, exposed to primary antibodies, including ASC (Abcam, ab283684, 1:1000), IL-1β (Abcam, ab254360, 1:1000), IL-18 (Abcam, ab207324, 1:1000), Pro-IL-18 (Proteintech, 10,663–1-AP, 1:1000), NLRP3 (Abcam, ab263899, 1:1000), IL-6 (Abcam, ab9324, 1 µg/ml), Sox4 (Abcam, ab70598, 1:500), JAK2 (Abcam, ab108596, 1:1000), pJAK2 (Abcam, ab32101, 1:1000), STAT3 (Abcam, ab68153, 1:1000), pSTAT3 (Abcam, ab267373, 1:1000) at 4℃ overnight, and secondary antibodies (Abcam) for 1 h. Finally, western blotting was developed after processing with ECL kit (Thermo scientific), and the brightness of each strip can be controlled by adjusting the exposure time.

Mdivi-1(mitochondrial division inhibitor-1) and TTC (2,3,5-triphenyltetrazolium chloride) were purchased from Sigma Aldrich (St. Louis, Missouri, MO, USA); BSA (bovine serum protein) was obtained from BOSTER (Boster Biological Technology, Wuhan, China, AR1006). Blots were incubated with antibodies against ATF4 (Proteintech, Rosemont, Pennsylvania, USA, 10835-1-AP), Parkin (CST, Boston, Massachusetts, USA, #4211), TOM20 (Proteintech, USA, 11802-1-AP), COX4I1 (Proteintech, USA, 60251-1-Ig), NLRP3 inflammasome (Affinity, Cincinnati, Ohio, USA, DF7438), pro-caspase-1 (ABclonal, Wuhan, China, A0964), cleaved caspase-1 (CST, USA, #67314), pro-IL-1β (ABclonal, China, A11370), cleaved IL-1β (Affinity, USA, AF4006), pro-IL-18 (Proteintech, USA, 10663-1-AP), cleaved IL-18 (R&D Systems, Minneapolis, Minnesota, USA, AF521) and β-actin (ABclonal, China, AC004), followed by the secondary antibodies conjugated to horseradish peroxidase anti-rabbit IgG (H + L) (ABclonal, China, AS014) and anti-mouse IgG (H + L) (ABclonal, China, AS003).
The primary antibodies used for the immunofluorescence analysis were against TOM20 (Proteintech, USA, 11802-1-AP) and COX4I1 proteins (Proteintech, USA, 60251-1-Ig). Brain sections were then incubated with the secondary antibodies goat anti-rabbit DyLight 488 (Abbkine, green, A23240) and goat anti-mouse DyLight 549 (Abbkine, red, A23310).