Cleaved casp3

To prepare whole-cell lysates, cells were washed twice with cold PBS and lysed in M-PER Mammalian Protein Extraction Reagent (product no. 78501, Thermo Fisher Scientific) supplemented with Pierce protease and phosphatase inhibitor cocktail (product no. A32961, Thermo Fisher Scientific). Tumor tissue samples were lysed in T-PER Tissue Protein Extraction Reagent (product no. 78510, Thermo Fisher Scientific) with above supplement. The automatic hand-operated OMNI-TIP Homogenizer (Omni International, Inc.) was used to homogenize the tumor tissues. Lysates from whole cells and tumor homogenates were then processed for SDS-PAGE Western blotting. The following antibodies were used: Phospho-MEK1/2 (S217/221; catalog no. 9154S), MEK1/2 (catalog no. 4694S), Phospho-AKT (S473; catalog no. 9271S), Phospho-AKT (T308; catalog no. 4056S), AKT (catalog no. 9272S), Phospho-Erk1/2 (catalog no. 4370S), Erk1/2 (catalog no. 4695S), Phospho-S6 Ribosomal Protein (Ser 235/236; catalog no. 4858S), S6 Ribosomal Protein (catalog no.2217S), cleaved-CASP-3 (catalog no. 9664), cleaved-PARP (catalog no. 9541) from Cell Signaling Technology; anti-β-ACTIN (catalog no A5441-.2ML from Sigma-Aldrich; KRAS (catalog no. OP24, from EMDMillipore), and RAF1 (catalog no. sc-7267, Santa Cruz Biotechnology), horseradish peroxidase–conjugated goat anti-GST antibody (catalog no. A190-121P from Bethyl Laboratories).

Mouse tissues and treated cells were lysed by RIPA (Solarbio® Life Science, R0010) that containing proteases and phosphorylases inhibitor cocktail. After BCA assay (Solarbio® Life Science, PC0020) for determining protein concentrations, 30 μg samples were loaded to a 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then underwent the electrophoresis under 80 V for 120 min. The gel was transmitted to a methanol activated polyvinylidene fluoride (PVDF) (Millipore®, Meck, 0.45 um, IPVH00010) membranesand blocking by 5% BSA at 4℃ for 2 h. The next day, the PVDF membrane were cut into different strips according to molecular weight of different protein like MLKL (Cell Signaling Technology™, CST37705), RIPK3 (Cell Signaling Technology™, CST95702), p-MLKL(Cell Signaling Technology™, CST37333), p-RIPK3(Cell Signaling Technology™, CST93654), LC3b II (Abcam, ab192890), p62 (Abcam, ab109012), cleaved Casp3 (Cell Signaling Technology™, 9664), GAPDH (Servicebio®, GB15002), Actin (Servicebio®, GB111364), citrullinated Histone H3 (H3cit) (Abcam, ab219407), and so on at 4℃ overnight. The membranes were washed with TBST for three times and incubated with GAR/GAM-HRP for 2 h at room temperature. At last, the membranes were washed and stained by ECL chemiluminescence.

Proteins were extracted from kidney tissues or HK-2 cells using RIPA buffer containing 4% cocktail proteinase inhibitors and then analyzed by western blotting. Equal amounts of protein were separated by SDS-polycrylamide gels and then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies against FABP4 (Abcam, Cambridge, MA, USA), Bax (Abcam, Cambridge, MA, USA), Bcl-2 (Abcam, Cambridge, MA, USA), cleaved-casp-3 (Cell Signaling Technology), GRP78 (Cell Signaling Technology), CHOP (Cell Signaling Technology), caspase-12 (Cell Signaling Technology, Beverly, MA, USA) and β-actin (Abcam, Cambridge, MA, USA) overnight at 4 °C followed by incubation with secondary antibodies (R&D Systems, MI, USA) for 1 h at room temperature. Finally, the proteins were developed with an enhanced chemiluminescence agent (Millipore Corporation, Boston, MA, USA). The signals were measured using an Odyssey Infrared Imaging System (Bio-Rad, ChemiDoc MP, mANUSC, Bio-Rad Laboratories Inc., Hercules, CA, USA) and quantified using the Image J program (National Institutes of Health, Bethesda, MD, USA). The ratio for the target proteins were normalized against β-actin.

After the desired treatment, cells were washed twice with cold PBS and harvested with a rubber scraper. Cell pellets were lysed and kept on ice for at least 30 min in a buffer (Beyotime Biotechnology, P0013) containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40 (Beyotime Biotechnology, P0013F), 50 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (Beyotime Biotechnology, ST506). The lysates were cleared by centrifugation, and the supernatant fractions were collected. Cell lysates were then separated by SDS-PAGE (Beyotime Biotechnology, P0012A) and subjected to western blot analysis with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Antibodies against the following were used for western blotting: SQSTM1 (Cell Signaling Technology, 5114S), LC3B (Abcam, ab63817), GAPDH (Cell Signaling Technology, 3683S), STAT3 (Abcam, ab68153), p-STAT3 (Abcam, ab76315), ACTB (Cell Signaling Technology, 12262S), PARP (Abcam, ab32138), cleaved PARP (Abcam, ab32064), CASP3 (Cell Signaling Technology, 9668T), cleaved CASP3 (Cell Signaling Technology, 9664S), KI67/PCNA (Cell Signaling Technology, 2586S), CREBRF (Santa Cruz Biotechnology, sc-133747), CREB3 (Abcam, ab42454), and ATG5 (Abcam, ab108327). GAPDH served as the loading control in all experiments involving hypoxic treatment due to the upregulation of ACTB under hypoxia.

The following primary antibodies were used: Lamp1 (Santa Cruz Biotechnology), LC3 (MBL International Corporation), p62 (MBL International Corporation), PARP 1(Cell Signaling Technology), PRKAA/AMPK (Cell Signaling Technology), phosphor-PRKAA/ AMPK(Cell Signaling Technology), Beclin1 (Cell Signaling Technology), ATG5 (Cell Signaling Technology), CASP9 (Cell Signaling Technology), CASP3 (Cell Signaling Technology) and Cleaved-CASP3 (Cell Signaling Technology). TBM (BP1415) was purchased from Phytopurify Biotechnology. 3-methyladenine (3-MA), chloroquine (CQ) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich. Doxorubicin (DOX, HY-15142A) and 5-fluorouracil (5-FU, HY-90006) were from MedChem Express.

Various antibodies from different vendors were used in this study.
Cell Signaling (Danvers, MA): PARP (9542); cleaved PARP [Asp214, 89 kDa fragment] (9545/94885); cleaved Casp3 [Asp175, 17 kDa fragment] (9661/9664); cleaved Casp7 [20 kDa fragment] (9492); phospho-H₂AX (S139) (9718); phospho-p53 (S15) (12571); total p53 for ChIP (32532); PUMA (14570); AIF (4642); cytochrome c oxygenase IV (COX IV) (4884); Lamin A/C (4777); phospho-c-Jun (S63) (2361); phospho-c-Jun (S73) (3270); total c-Jun (9165); total c-Fos (2250). Millipore (Ontario, Canada): phospho-ATM(S1981) (05-740); Sp1 for ChIP (07-645); p21—mouse samples (188224); Milli-Mark^TM Pan-Neuronal Marker (MAB2300). BD Biosciences (San Jose, CA): p21—rat samples (556430). Sigma-Aldrich (St. Louis, MO): β-actin: A1978. Santa Cruz (Dallas, TX): cytochrome c (sc-13560). Enzo (Farmingdale, NY): α-Fodrin or αII-spectrin (a subunit of Fodrin, also known as “brain spectrin”)^{41 (link)}: BML-FG6090. Abcam (Cambridge, MA): phospho-p53 (ab1431).