Systematic Knockdown of Key Autophagy Genes

For siRNA transfection as described previously [15 (link)], cells were reversely transfected with RNA iMAX (Life Technologies; 13778–150) and 5 nM scramble siRNA (Life Technologies, 12935–112), kinome siRNA library (2127 siRNA for 709 gene, A30079, Thermo Fisher Scientific), ATG5 (GE Healthcare Dharmacon, 9474), ATG7 (Life Technologies, s20652), BECN1 (Life Technologies, 4392420), ULK1 (Dharmacon, 8408), or ERBB2 (Life Technologies, 4390824;) for 72 h. For shRNA infection, 8XARE reporter plasmid, a packaging and VSV-G expressing envelope plasmid, were transfected into HEK293T cells using the transfection reagent, Lipofectamine 2000 (Life Technologies, 11668–027) for two days to achieve an infection. Cells were then harvested for further experiments or to confirm knockdown efficiency via immunoblotting.