Knockdown of RORA and RORC in Cell Lines

Cells were incubated at 37 °C in a humidified chamber at 5% CO₂. Lung cancer cells (A549, H441, and H358) were cultured in RPMI-1640 medium (GenDEPOT, TX, USA) and other cells (HPNE-P2M, HPNE-Kras, PNAC1, MCF7, MDA-MB-231, MDA-MB-468, DB7, and BT549) were cultured in DMEM medium (GenDEPOT). All media were supplemented with 10% FBS medium (GenDEPOT) and 1% penicillin–streptomycin medium (GenDEPOT).
Lipofectamine RNAiMAX (Invitrogen, CA, USA) was used for RNA-oligonucleotide transfection, whereas plasmid transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. All oligonucleotides were purchased from Sigma (MO, USA). The sequence of each gene is shown in follows. RORA and RORC siRNA are siRORA: 5′-CAAGAUCUGUGGAGACAAAdTdT and siRORC: CGAGGATGAGATTGCCCTCTAdTdT, respectively. For negative control, MISSION® siRNA Universal Negative Controls #2 (Sigma, MO, USA) was used.
RORA and RORC CRISPR knockdown cells were generated as previous described [40 (link)]. Briefly, sgRNAs were designed by using the CPRISRdirect software (https://crispr.dbcls.jp/). MDA-MB-231 cells were transfected with RORA and RORC sgRNA constructs and selected by puromycin. Colonies were expanded and validated for knockdown efficiency. Experiments were performed by using cells at early passages (before p5).