Psicor pgk puro

The short hairpin RNA (shRNA) specifically against BRAF and the scramble control shRNA were cloned into the lentiviral vector pSicoR-PGK-puro (#12084, Addgene, Cambridge, MA)^{52 (link)}. A pLKO.1-puro based lentiviral vector expressing shRNA against TERT (#TRCN0000240466), FOS (#TRCN0000016007) and MYC (#TRCN0000039642) were purchased from Sigma-Aldrich and the pLKO.1-puro vector with scramble shRNA was purchased from Addgene (plasmid #1864). To generate lentiviral particles, the lentiviral shRNA-expressing vector with the packaging plasmid PSPAX2 and the VSV-G envelope protein-coding plasmid pMD2.G were co-transfected to HEK293T cells using Lipofectamine 3000 (Invitrogen) and the supernatant was harvested 48 h after transfection. To generate cell lines with stable knockdown of BRAF, TERT, FOS or MYC, cancer cells were exposed to the above lentivirus-containing supernatant for 24 h in the presence of 8 μg ml⁻¹ polybrene (Millipore, Billerica, MA) and selected by 2 μg ml⁻¹ puromycin (Sigma-Aldrich) for 2 weeks. The stable transfection cell pools were confirmed by western blotting analysis of the proteins of interest.

Retroviral vectors pBABE-puro (Addgene Inc. Cambridge, MA, USA) containing the full-length wild-type BRAF and BRAF V600E cDNA were used for stable overexpression of BRAF. Lentiviral pSicoR-PGK-puro vectors (Addgene Inc.) encoding hairpin RNA sequences were used to achieve stable knockdown of BRAF. The pLKO.1-shWIPF1 (Sigma) was used to achieve knockdown of WIPF1. To generate lentiviral particles, human embryonic kidney 293T cells were co-transfected with the viral vector and compatible packaging plasmid mixture (psPAX2 and pMD2.G, Addgene Inc.) using Lipofectamine 3000 (Invitrogen) following the manufacturer's instructions. K1, OCUT1, FTC133 and WRO cells were exposed to virus-containing supernatant for 16 hours in the presence of 8 μg/mL polybrene (Sigma). After 7-10 days of selection with puromycin (Sigma), cells were serum-starved (0.5% FBS) and harvested 24 hours later in RIPA lysis buffer (Santa Cruz Biotechnology). The expression of BRAF, p-ERK, and WIPF1 was examined by Western blotting.