CRC cell lines (HT29, HCT8, LOVO, HCT116, SW620, SW420) and normal human colonic epithelial cells (FHC) were purchased from the American Type Culture Collection (Manassas, VA, USA), and cultured in DMEM basic medium with 10% fetal bovine serum (FBS, Thermo Fisher Scientific/Gibco, Gaithersburg, MD, USA) and 100 U/mL penicillin as well as 100 μg/ml streptomycin.
For transient transfection, SW620 cells were transfected with 100 nM siRNA targeting B7-H4 (si-B7-H4) and LOVO cells were transfected with 6 µg pcDNA3.1-B7-H4 expression plasmid or their controls using Lipofectamine 2000 (Invitrogen, California, USA). For stable knockdown, SW620 cells were infected with recombinant lentiviruses containing short hairpin RNA (shRNA) targeting human B7-H4 (SW620-shB7-H4) or negative control (SW620-sh-NC) construct. The recombinant lentivirus carrying shRNA of B7-H4 or negative control was prepared by Genechem Co., Ltd., (Shanghai, China), and lentiviral infection was carried out according to the manufacturer’s instructions. 24 h post infection, cells were selected by 1.0 μg/mL puromycin for one week to eliminate uninfected cells. For the drug treatment, 0.1 μM capmatinib (INC280, Novartis) was added to the culture medium and incubated for 24 hours in LOVO cells with B7-H4 overexpression.
For transient transfection, SW620 cells were transfected with 100 nM siRNA targeting B7-H4 (si-B7-H4) and LOVO cells were transfected with 6 µg pcDNA3.1-B7-H4 expression plasmid or their controls using Lipofectamine 2000 (Invitrogen, California, USA). For stable knockdown, SW620 cells were infected with recombinant lentiviruses containing short hairpin RNA (shRNA) targeting human B7-H4 (SW620-shB7-H4) or negative control (SW620-sh-NC) construct. The recombinant lentivirus carrying shRNA of B7-H4 or negative control was prepared by Genechem Co., Ltd., (Shanghai, China), and lentiviral infection was carried out according to the manufacturer’s instructions. 24 h post infection, cells were selected by 1.0 μg/mL puromycin for one week to eliminate uninfected cells. For the drug treatment, 0.1 μM capmatinib (INC280, Novartis) was added to the culture medium and incubated for 24 hours in LOVO cells with B7-H4 overexpression.