Human donor–derived skin fibroblasts were obtained with informed consent, approved by ethics committee, and have been described previously (3237). Antibodies and key reagents used in the study are listed in tables S1 and S2. Cell lines (healthy donor–derived control, which we call C12 cells here, HeLa, HEK293E, HEK293T, and L929) were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) plus penicillin and streptomycin, sodium pyruvate, and 10% heat-inactivated fetal bovine serum (all from Sigma-Aldrich). Retrovirally and lentivirally transduced cell lines were cultured in the same medium plus puromycin (2 μg/ml; Sigma-Aldrich). All cell lines were maintained at 37°C and 5% CO2 and were tested to be mycoplasma-negative (LookOut Mycoplasma PCR Detection Kit, Sigma-Aldrich).

Unless otherwise specified, cells were treated with poly(I:C) (25 μg/ml), PAM2CSK4 (5 μg/ml), or ultrapure Escherichia coli O111:B4 lipopolysaccharide (5 μg/ml) for 20 hours or as indicated. The following inhibitors were used: Ac-YVAD-fmk (50 μM), Ac-IETD-fmk (50 μM), Baf-A1 (100 nM), epoxomicin (5 μM), necrostatin-1 (25 μM), Torin 1 (250 nM), and z-VAD-fmk (50 μM). Cells were passaged by trypsinization and usually cultured for ~4 to 10 weeks for experiments. HeLa cells were plated at a density of 7.5 × 104 per well in 96-well plates for ELISA and 2 × 105 per well in 24-well plates for immunoblots. C12 cells were plated at a density of 1.75 × 104 per well in 96-well plates for ELISA, LDH, and PI assays in phenol red–free media and 7 × 104 per well in 24-well plates for immunoblots.

For starvation of all amino acids, cells were washed with warm phosphate-buffered saline (PBS) before incubation in serum- and amino acid–free Earle’s balanced salt solution (Gibco) for 45 min (skin fibroblasts) or 2 hours (HeLa) and stimulated for 15 min by addition of 1× amino acid mixture containing all amino acids at a final concentration that matched RPMI (from a 50× solution plus glutamine, both from Sigma-Aldrich). For single amino acid starvation and restimulation experiments, cells were washed with PBS and incubated in serum-free RPMI lacking leucine or arginine for 60 min (skin fibroblasts) or 4 hours (HeLa) and stimulated for 30 min by adding leucine or arginine, respectively. After stimulation, the final concentration of amino acids in the media was the same as in RPMI. For glucose starvation and restimulation, cells were starved of glucose for 80 min (skin fibroblasts) or 6 hours (HeLa) and stimulated with 25 mM glucose for 30 min. For starvation experiments in Δp62/Flagp62TEV+TEV-T2A-GFP cells, doxycycline was added for 24 hours (1 μg/ml), and then cells were left to recover in the absence of doxycycline overnight. Starvation experiments were performed the following day as described above. Where indicated, Ac-IETD-fmk (50 μM) was added 30 min before starvation and replenished during starvation. Immunoblotting, ELISAs, and cell death assays were described before (53). Staurosporine (1 μM) was used for 6 hours and actinomycin D (2 μM) for 20 hours. Live time-lapse images were taken using a Cytation 1 cell imaging multi-mode reader (BioTek Instruments). Necroptotic cell death was induced by treatment with TNF (10 ng/ml) combined with cycloheximide (5 μg/ml), birinapant (100 nM), and zVAD-fmk (50 μM) for 20 hours. When used, necrostatin-1 (25 μM) was added at the same time.

Cell lysates were prepared in radioimmunoprecipitation assay buffer [120 mM tris (pH 8.0), 300 mM NaCl, 2% NP-40, 1% Na-deoxycholate, 2 mM EDTA] supplemented with complete protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride (PMSF), and phosphatase inhibitor tablets and then mixed with Laemmli buffer containing 5% 2-mercaptoethanol. Proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) using tris-glycine buffer systems and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories) through semidry transfer. For p62 immunoblots, typically, the primary antibody was incubated in 2% fat-free milk in PBS–Tween 20 (PBST) (4°C overnight) and secondary antibody in 5% fat-free milk in PBST (1 hour at room temperature). Immunoblots were routinely developed with Clarity Western enhanced chemiluminescence (ECL), and ECL prime substrate was used for the detection of poorly expressed proteins or cleaved caspase subunits. Immunoblot quantification used images from independent experiments acquired on a ChemiDoc MP (Bio-Rad) and analyzed using Image Lab software (Bio-Rad Laboratories).

For IL-6 ELISA from resting cells, supernatants were collected 20 hours after plating. When used for 72 hours, Torin 1 and Ac-IETD-fmk were replenished daily. Human IL-6 ELISA kit was from eBioscience (#88-7066).

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