Hcclm3

HCC cell lines (SK-HEP-1, HCCLM3, MHCC97L, Huh7 and Hep3B) and human liver cell line LO2 were from BeNa Culture Collection (Chaoyang, Beijing, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with fetal bovine serum (10%, FBS, GIBCO, Grand Island, NY, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin in a condition of 37 °C and 5% CO₂.

The human hepatocytes and liver cancer cells including Huh-7, Hep3B2, Li7, and HCCLM3 were obtained from Bena Culture Collection (Beijing, China). These cells were cultured in DMEM medium supplemented with 10% FBS at 37°C atmospheres with 5% CO₂.

HCC cell lines (HCCLM3 (BNCC338460), MHCC97-H (BNCC359345), and HepG2 (CL-0103)) and hepatic epithelial cells (THLE-2 (BFN60808733)) were obtained from BeNa Culture Collection (Suzhou, China), Procell (Wuhan, China) and Qingqi Biotechnology Development Co., Ltd (Shanghai, China). These cell lines were cultured in complete medium of Dulbecco’s modified Eagle’s medium. The HepG2 and HCCLM3 cells were transfected in 6-well plates (NEST Biotechnology, Wuxi, China) using Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions. The corresponding small-interfering RNA (siRNA) sequences are listed in Table S2. HCC cells were subjected to pyroptosis induction using LPS (1 μg/mL) and ATP (100 μM) as in previous studies (25 (link)).

Three HCC cell lines, including conventional hepatocellular carcinoma cell line (Huh7) and two highly metastatic HCC cell lines (MHCC97‐H and HCCLM3) were acquired from BeNa Culture Collection (Beijing, China). The three cells were grown in Roswell Park Memorial Institute‐1640 (RPMI‐1640) medium (Gibco, Carlsbad, CA, USA) added with 10% fetal bovine serum (FBS; Gibco), 100 units/mL penicillin/100 μg/mL streptomycin in a 37℃ humidified atmosphere with 5% CO₂. HCC cells were treated with increased concentrations (0 μmol/L, 5 μmol/L, 25 μmol/L, or 50 μmol/L) of Propofol (Sigma, St. Louis, MO, USA) for 24 hours.

The HCC cell lines including HepG2, Huh‐7, HCCLM3, SNU449, SNU475, HepaRG and human normal hepatic cell line HL‐7702 were gained from BeNa Culture Collection (Beijing, China). HepG2, Huh‐7 and HCCLM3 cell lines were maintained in high‐glucose DMEM medium (Invitrogen, Carlsbad, CA, USA) with 10% foetal bovine serum (FBS, Invitrogen, CA, USA). HL‐7702, SNU449, SNU475 and HepaRG cells were cultured in RPMI‐1640 medium (GIBCO, Carlsbad, CA, USA) with 10% FBS (Invitrogen).

HCC Cells (SMMC-7721, Hep3B, HCCLM3, HepG2, MHCC97H, Huh7, QSG-7701), and LO2 cells (as control cells) were bought from Bena Culture Collection (Kunshan, Jiangsu, China). They were cultured by RPMI-1640 media (with 10% fetal bovine serum). The cell culture condition was: 37 ˚C, 5% CO₂. The siRNAs (siRNA-control, siTNA-STAT1, siGOLM1-1, siGOLM1-2), miR-653 mimics and inhibitors were purchased from JiMa Biological corporation (Suzhou, Jiangsu, China). The overexpressing plasmids, including pcDNA3.1-STAT1, pcDNA3.1-ZFPM2-AS1, and pcDNA3.1- GOLM1, were Genetong Biological corporation (Xiamen, Fujian, China). The cell transfection was conducted using Lipofectamine 2000 reagent kits (GuangHua Biotech, Changsha, Hunan, China) in accordance with the kits’ protocols.

The LIHC cell lines HCCLM3 and MHCC97-H and the hepatic epithelial cell line THLE-2 were acquired from BeNa Culture Collection. The HepG2, U-251 MG, and LN229 cell lines were obtained from Procell and cultured according to the manufacturer’s instructions. Transfection was carried out in 6-well plates (NEST Biotechnology) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol in HepG2, HCCLM3, U-251 MG, and LN229 cells. The siRNA used in this study was synthesized by GenePharma. Supplementary Table S1 lists the sequences of the siRNAs used in this study. The Western blot experimental steps were described in a previous study (28 (link)). The antibodies used in this study were anti-ORC6 (Proteintech, 17784-1-AP, 1:1000) and anti-alpha tubulin (Proteintech, 11224-1-AP, 1:5000).