H295R human adrenocortical carcinoma cells were cultured in DMEM/Nutrient F-12 Ham supplemented with 10% calf bovine serum, 100 U penicillin, 0.1 mg/mL streptomycin, 0.4 mmol/L l -glutamine, and insulin-transferrin-sodium selenite medium at 37°C in 5% CO2 as previously described.5 (link) Gene overexpression was performed using lipid-mediated cell transfection Lipofectamine 3000 (Thermo Fisher). Cells were transfected with GFP-tagged ANO4 (EX-Mm36875-M29, EX-W1648-M29, EX-I1781-M29, EX-T8891-M29, Genecopoeia), or empty vector control. At 24 hours, H295R cells were serum deprived in unsupplemented medium for 12 hours, and further drug treatments were carried out with 24 hours incubation from this point.
Gene silencing was achieved using DharmaFECT 1 lipid transfection reagent (Dharmacon). Cells were silenced using either ON-TARGETplus nontargeting small interfering RNA as control or SMART pool: ON-TARGET plus ANO4 small interfering RNA (Dharmacon; 50 nmol/L). Cells were harvested for analysis of mRNA and protein expression after 48 hours.
Gene silencing was achieved using DharmaFECT 1 lipid transfection reagent (Dharmacon). Cells were silenced using either ON-TARGETplus nontargeting small interfering RNA as control or SMART pool: ON-TARGET plus ANO4 small interfering RNA (Dharmacon; 50 nmol/L). Cells were harvested for analysis of mRNA and protein expression after 48 hours.