Pepstatin a

ATCC supplied all human cancer cells and normal mouse kidney (TCMK-1) cells (Manassas, VA), and Lonza supplied normal human mesangial cells (Basel, Switzerland). All cell lines tested negative for mycoplasma contamination. The lines were authenticated by standard morphologic examination using microscopy. Cells were cultured in DMEM containing 10% FBS and 100 μg/mL gentamycin. R&D system supplied z-VAD-fmk and TNF-α (Minneapolis, MN), and Biomol supplied curcumin (Plymouth Meeting, PA). LKT Labs (St. Paul, MN) and Selleckchem (Huston, TX) supplied Kahweol and PP242, respectively. Calbiochem supplied Quercetin, Luteolin, 2-aminoethosxydiphenyl borate (2-APB), and EGTA-AM (San Diego, CA). Enzo Life Sciences (Plymouth Meeting, PA) and Cayman Chemical (Ann Arbor, MI) supplied pepstatin A and E64D and, respectively. Sigma Chemical Co. supplied other chemicals (St. Louis, MO). We provided information for used antibodies in Supplementary Table 1. Human Akt1 cDNA (Upstate Biotechnology, Lake Placid, NY) was cloned into pcDNA3.1-Myc/His vector. pRK5 vector was purchased from Clontech Laboratories, Inc. (Mountain View, CA). pRK5-myc-Raptor (Addgene plasmid # 1859) and pRK5-myc-Rictor (Addgene plasmid # 1859) were a gift from David Sabatini [52 (link)]. Santa Cruz Biotechnology supplied the siRNA (Santa Cruz, CA).

Immunoprecipitation was performed as previously described (64 (link)). Caki-2 cells (1 × 10⁷) were harvested and lysed in 2 ml of buffer A (20 mM Hepes pH 7.9, 25 mM KCl, 0.1% v/v Nonidet P-40, 10% v/v glycerol) plus 1:1000 leupeptin, pepstatin A, and aprotinin protease inhibitors (Cayman Chemical) and centrifuged at 600g for 10 min. The nuclei were then resuspended in 250 μl of immunoprecipitation (IP) buffer (25 mM Tris pH 8.0, 300 mM NaCl, 1% v/v Nonidet P-40, 1 mM EDTA, plus protease inhibitors) and rotated at 4 °C for 30 min. The extracts were cleared by centrifugation at 21,000g for 30 min. The cleared extract was precleared with normal immunoglobulin G (IgG)-conjugated protein A/G magnetic beads (Pierce) for 20 min. One microgram of specific IgG was used per 0.2 mg lysate for immunoprecipitation. After overnight incubation, immunocomplexes were captured using protein A/G magnetic beads following a 2-h incubation. The beads were washed twice in chromatin IP buffer and three times in high stringency wash buffer (20 mM Hepes pH 7.9, 500 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA). The proteins were eluted in 1× lithium dodecyl sulfate loading dye (Thermo Fisher Scientific) by heating at 70 °C for 10 min. Samples were heated at 95 °C for 10 min then loaded onto a 4 to 12% SDS-polyacrylamide gel (Invitrogen) for immunoblotting.

Antibodies for PARP (9532, 1:1000), ULK1 (8054, 1:1000), p62 (5114, 1:1000), AIF (5318, 1:1000) and Lamin B (12586, 1:1000) were purchased from Cell Signaling Technology (MA, USA). TFG (ab156866, 1:10,000) and GSDMD (ab210070, 1:1000) were from Abcam. Caspase-1 (AG-20B-0048-C100, 1:1000) were purchased from AdipoGen (CA, USA). HA (12CA5, 1:1000 for IP) and HA-HRP (3F10, 1:5000) were from Roche Life Science (Switzerland). Flag (F3165, 1:500 for IP) and Flag-HRP (A8592, 1:5000), LC3B (L7543, 1:1000) and β-actin (A2228, 1:10,000) were from Sigma–Aldrich (MO, USA). Ub (P4D1, 1:1000), TRAF3 (H-122, 1:1000), Tubulin (SC-8035, 1:1000), and rabbit IgG (sc-2027, 1:1000) were from Santa Cruz Biotechnology (CA, USA). COX4 (ARG66326, 1:1000) were purchased from Arigo Biolaboratories. V5 (R96025, 1:500 for IP) and V5-HRP (R96125, 1:5000) were from Thermo Fisher Scientific (MA, USA).
MitoSox™ Red Mitochondrial Superoxide Indicator (M36008), SYTOX™ Blue Dead Cell Stain (S34857), and SYTOX™ Red Dead Cell Stain (S34859) were purchased from Thermo Fisher Scientific (MA, USA). LPS, Nigericin, Z-VAD-FMK (Z-VAD), Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma–Aldrich (MO, USA). Necrostatin-1 (NEC-1) was purchased from ApexBio (Texas, USA). Cycloheximide (CHX), E-64d, and Pepstatin A were purchased from Cayman Chemical (Michigan, USA).