Sox 2 sirna

SOX2-silenced A375-M6 cells were obtained by siRNA transfection with Sox-2 siRNA (sc-38408, Santa Cruz Biotechnology, Dallas, Texas, USA) or control siRNA-A (sc-37007, Santa Cruz Biotechnology), according to manufacturer’s instructions. SOX2 silencing in SSM2c cells was obtained by lentiviral transduction. Lentiviruses were produced in HEK-293 T cells. Lentiviral vectors used were pLKO.1-puro (LV-c) (Open Biosystems, Lafayette, CO, USA) and pLKO.1-puro-shSOX2–1 (LV-shSOX2–1) targeting the 3′ untranslated region of SOX2 (targeting sequence 5’-CTGCCGAGAATCCATGTATAT-3′) as previously reported [13 (link)]. SOX2 overexpression in 501-Mel cells was obtained by retroviral transduction. Retroviruses were produced in HEK-293 T cells. Retroviral vectors used were generated by co-transfection of 1 μg pBABE (Addgene, Cambridge, MA, USA, #1764) or pBABE-SOX2 (cloned into the BamHI/SalI restriction sites of pBABE vector using the following primers: SOX2-F 5’-ATGTACAACATGATGGAGACGG-3′ and SOX2-R 5’-TCACATGTGTGAGAGGGGC-3′), 0.9 μg pUMVC packaging plasmid (Addgene, #8449) and 0.1 μg pCMV-VSV-G envelope (Addgene, #8454).