CD274 and melanoma: To investigate nicotine-induced α9-nAChR and PD-L1 upregulation, we performed western blotting to detect α9-nAChR and PD-L1 protein levels in the A375, A2058 and MDA-MB 435 melanoma cells treated with nicotine at final concentrations of 0.001–1 μm for 24 h or at a concentration of 1 μm for 12–72 h.