Stealth rna interference rnai

HCC cell lines were obtained from the Japanese Cancer Research Resources Bank (JCRB) Cell Bank (Osaka, Japan) and KAC (Kyoto, Japan), and were confirmed to be free from mutation in ARID1A and PD‐L1 genes using the database of the Cancer Dependency Map (https://demap.org/portal/), Hep3B (Cell ID ECACC 86062703; Depmap ID ACH‐000625; http://demap.org/potral/cell_line/ACH‐000625?tab=mutaion), HuH7 (Cell ID JCRB 0403; Depmap ID ACH‐000480; http://demap.org/potral/cell_line/ACH‐000480?tab=mutaion), and PLC/PRF5 (Cell ID JCRB 0406; Depmap ID ACH‐001318; http://demap.org/potral/cell_line/PLCPRF5_LIVER?tab=mutaion). Cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 1% penicillin‐streptomycin at 37℃ at 10% CO₂. To generate ARID1A knockdown cells, transient gene suppression was performed in Hep3B and HuH7 HCC cells using Stealth RNA interference (RNAi; Thermo Fisher Scientific, Waltham, MA). Cells were prepared in complete growth medium without antibiotics to obtain a 500‐µL suspension containing 25,000 cells (30%‐50% confluent 24 hours after plating). Then, reverse transfection was performed using 10 nM Stealth RNAi with Opti‐MEM I Reduced Serum Medium and Lipofectamine RNAiMAX for 48 hours at 37°C in a CO₂ incubator.

HuH7 and Hep3B cells were obtained from the American Type Culture Collection (Manassas, VA). HuH7 and Hep3B were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37ºC in 10% CO₂. Antibodies against phosphorylated NRF2 (P‐NRF2), NRF2, PD‐L1, and HIF1α were purchased from Abcam (Cambridge, United Kingdom). β‐actin antibody was obtained from Sigma‐Aldrich (St. Louis, MO). Stealth RNA interference (RNAi; Thermo Fisher Scientific, Waltham, MA; GE Healthcare Dharmacon, Lafayette, CO) was used to knock down HIF1A.