Pmd2 g vector

mCherry-Lifeact was purchased from Addgene (#54491; Cambridge, MA), and pEGFP-Myo10 was previously described^{5 (link)}. To knockdown mouse MYO10 expression, we used the pLKO.1-TRC lentiviral vector (MISSION shRNA, Sigma-Aldrich) containing shRNA against mouse MYO10 (shRNA-1;TRCN0000110606, shRNA-2;TRCN0000110608, and shRNA-3;TRCN0000379126). A non-target shRNA vector (SHC016; Sigma-Aldrich) was used as a control for the shRNA experiments. For lentiviral packaging, psPAX2 and pMD2.G vectors (Addgene plasmids #12260, #12259) were co-transfected with the pLKO.1 into HEK293T cells with X-tremeGENE HP (Sigma-Aldrich) according to the manufacturer’s instructions. Virus-containing medium was harvested 72 hrs after transfection and filtered with 0.45 μm filters (EMD Millipore, Billerica, MA). Melb-a or B16F1 cells were infected with culture supernatants from HEK293T cells at a 1:6 dilution in complete culture medium. Cells were passed after 24 hrs and selected with 4 μg/ml puromycin for 14 days.

For the gene repression experiments, the genes were downregulated via lentivirus transfection with specific shRNAs. The lentiviral vector pLKO.1-TRC was used to construct the shRNA vectors. The DNA fragments encoding shRNAs targeting specific genes or a non-specific gene (Con shRNA) were synthesized by Genewiz (Beijing, China) and inserted into the Age I and EcoR I site of the pLKO.1-TRC vector and verified by DNA sequencing. The newly constructed lenti-shRNA vectors were subsequently co-transfected into 293TN cells (System Biosciences, Mountain View, CA) with the indicated second-generation packaging systems (psPAX2 and pMD2.G vectors, Addgene, Cambridge, MA) using Chemifect transfection reagents (Fengrui Biotechnology, Beijing, China). The lentivirus-containing supernatant was harvested after 48–72 hours of transfection and filtered through a 0.22-μm filter. Transduction was performed in the presence of 10 μg/mL polybrene for 48–72 hours, and gene knockdown efficiency was verified by western blotting. DNA sequences targeting the following specific genes were inserted into the lenti-shRNA vectors:
mouse RIP1 (5′-GCATTGTCCTTTGGGCAAT-3′), mouse RIP3 (5′-GCTGAGTTGGTAGACAAGA-3′), mouse caspase 8 (5′-GAATGGAACCTGGTATATT-3′) and mouse TRADD (5′-GCAAAGACCCTCTAAGTACCCGGAC-3′).