Ch25h

H9C2 cardiomyocytes were lysed in iced RIPA buffer (Solarbio, China) containing protease inhibitors for 30 min and centrifuged at 13523 g, 4°C for 10 min to collect the supernatants. The protein concentration was measured by the BCA protein assay kit (Solarbio). Proteins were then separated in sodium dodecyl sulfate (SDS) polyacrylamide gel by electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). Membranes were blocked with tris-buffered saline with tween 20 (TBST) buffer containing 5% nonfat milk and then incubated with primary antibodies against GAPDH (1:1000, Abcam, UK), Caspase-1 (1:1000, Abcam), NLRP3 (1:1000, Proteintech, China), GSDMD (1:1000, Biorbyt, UK), IL-1β (1:1000, Beyotime, China), and CH25H (1:500, Santa Cruz, USA) overnight at 4°C. After washing with TBST buffer three times, membranes were incubated with secondary antibody (Cell Signaling Technology, USA) for 2 h. The specific protein bands were detected using the Odyssey CLx imaging system (LI-COR, USA).

Standard hematoxylin/eosin staining was performed on 5 μm paraffin-embedded tissue sections after deparaffinization. For UCP1 staining, 5 μm paraffin-embedded tissue sections were blocked with 2.5% normal goat serum in PBST (Phosphate-buffer saline with 0.1% Tween 20) for 1 h at room temperature following deparaffinization. UCP1 primary antibody (1:50, Sigma-Aldrich) was applied overnight at 4 °C. The following day, secondary antibody against rabbit (SignalStain Boost IHC, Cell Signaling) was applied for 1 h at room temperature and developed with DAB kit (Vector Laboratories) according to the manufacturer’s instruction. For CH25H and perilipin staining, 5 μm paraffin-embedded tissue sections were incubated overnight at 4 °C with CH25H (1:50, Santa Cruz Biotechnology) or perilipin (undiluted, OriGene) primary antibody and stained with Tyramide SuperBoost Kit (Thermo Fisher) according to manufacturer’s instruction. Nucleus counterstaining was performed with NucRed 647 (Thermo Fisher).