Cy5582

Cells were lysed in RIPA lysis buffer (Beyotime) containing 1% phosphatase and protease inhibitors at 4 °C for approximately 30 min. Protein (30 μg) was separated by 10% SDS-PAGE (PAGE-Gel Fast Preparation Kit, EpiZyme) and transferred onto PVDF membranes (Pierce Biotechnology). Protein bands were blocked with 5% nonfat milk at room temperature for 2 h and incubated with the indicated primary antibody overnight at 4 °C and the appropriate secondary antibody for 2 h. ECL reagents (Abcam) were used for exposure. The following antibodies and dilutions were used: AIM2 (ab93015, Abcam, 1:1000), BCL2A1 (CY5582, Abways, 1:1000), p44/42 MAPK (137F5, CST, 1:1000), c-Myc (E5Q6W, CST, 1:1000), α-tubulin (AF0001, Beyotime, 1:1000), NF-κB Pathway Sampler Kit (#9936, CST, 1:1000), Phospho-Erk1/2 Pathway Sampler Kit (#9911, CST, 1:1000), IL-1β (#12703, CST, 1:1000), cleaved IL-1β (#83186, CST, 1:1000) and Caspase-1 (#3866, CST, 1:1000).

Paraffin-embedded tissue sections were processed according to standard pathologic procedures. BCL2A1 staining scores were multiplied by the staining intensity (0 for no staining, 1 for weak, 2 for clear and 3 for strong) and staining average (1 for 1-20%, 2 for 20-50%, and 3 for 50% above). The AIM2 IHC score was based on the staining intensity (1 for no staining, 2 for faint and weaker than interstitial cells, 3 for clear and similar to interstitial cells, 4 for yellow-brown and stronger than interstitial cells and 5 for extremely strong). The antibodies and dilutions were as follows: AIM2 (ab93015, Abcam, 1:250) and BCL2A1 (CY5582, Abways, 1:100). The cut-off value was calculated using X-Tile software (version 3.6.1) as previously described 25 (link). In our cohorts, the cut-off values of BCL2A1 and AIM2 IHC scores were both 2 points. A BCL2A1 expression score of 3-9 points and an AIM2 expression score of 3-5 points were regarded as high expression.