For stable gene silencing, 22 base oligonucleotides (first base mismatch plus 21-mer sense and 22-mer antisense without mismatches) were cloned in the optimized miR backbone (77, 78) in pMXCMV-YFP vectors as described previously (53). Antisense 22-mer sequences used were the following: p62 (targeting the 3′UTR), 5′-TTAACACAACTATGAGACAGAA-3′ (from TRCN0000430110); RIPK1, 5′-TTATCCGTCAGACTAGTGGTAT-3′; ATG7, 5′-ATGGAGAGCTCCTCAGCAGGCG -3′ (from TRCN0000007584); nontargeting control LacZ, 5′-TCACGACGTTGTAATACGACGT-3′ (TRCN0000072226).

Transient RNAi used ON-TARGETplus siRNA reagents from Dharmacon. Nontargeting control (Dharmacon) served as negative control. siRNA pools were transfected in cells using Viromer BLUE (Lipocalyx) or TransIT-X2 reagent (Mirus) following the manufacturer’s protocol. Briefly, C12 and HeLa cells were seeded the day before transfection at 2 × 104 per well and 4 × 104 per well, respectively, in 24-well plates. For C12 cells, 50 nM siRNA with 0.5 μl of Viromer was used per well, and 25 nM siRNA and 0.25 μl of Viromer were used for HeLa and HEK293T cells. When using the TransIT-X2 reagent, 20 nM siRNA and 1.5 μl of reagent were used per well. Media were changed 48 hours after transfection, and experiments were performed the following day. Silencing efficiency was assessed by immunoblotting. siRNA sequences are presented in table S3.

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