2 dg d8375

Ethanol (E7023) and 2DG (D8375) were from Sigma-Aldrich (St Louis, MO, USA). Erlotinib HCl (OSI-744) (S1023) was from Selleckchem (Houston, TX, USA). The following antibodies were used: monoclonal rabbit anti-HK1 (1:1000) (2024, Cell Signaling Technology (Danvers, MA, USA)), monoclonal rabbit anti-EGFR (1:1000) (ab52894, Abcam, (Cambridge, MA, USA)), polyclonal rabbit anti-Caspase-3 (1:1000) (#9662, Cell signaling Technology (Danvers, MA, USA)), monoclonal rabbit anti-Akt (1:1000) (#9272, Cell signaling Technology (Danvers, MA, USA)), monoclonal rabbit anti-phospho-Akt (Ser473) (#9271, Cell signaling Technology (Danvers, MA, USA)) and monoclonal mouse anti-β-actin (1:5000) (A5316, Sigma-Aldrich (St Louis, MO, USA)).

Melanoma cell lines as well as the normal human melanocytes HEMn-LP cells were all purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in DMEM or McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) and maintained in an incubator at 37 °C with 5% CO₂. Transfection of siRNA-control, siRNA-CCHE1, pcDNA-Flag-LDHA, or pcDNA-Flag-LDHA Y10F was performed with Polyethylenimine Linear (PEI, MW 40000, Sino Biologicals, Beijing, China) according to the manufacturer’s instructions. The cisplatin (232,120), dacarbazine (D2390), and 2-DG (D8375) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and stored at 4 °C (RT). The dacarbazine resistant A375 and G-361 cells were derived from the parental cells through 2 cycles of dacarbazine treatment with lethal dose that kills 90% of cells. After the screening, cells were cultured in fresh medium with10% FBS.