Cleaved caspase 9

Cells or frozen mouse eye sections were fixed in 4% paraformaldehyde for 15 min, washed in PBS, and blocked in ICC buffer with 5% goat or rabbit serum for 30 min at 4°C. Primary antibody incubation occurred overnight at 4°C. Secondary antibody incubation lasted 1 h at room temperature. Slides were mounted in Vectashield fluorescent media (Vector Labs) and imaged with an Olympus FV1000 Confocal Scanning Scope. Primary antibodies: Cleaved Caspase-3 diluted 1∶200 (Cell Signaling Technology, Danvers, MA), Cleaved Caspase-9 diluted 1∶100 (Santa Cruz), and NF-κB p65 diluted 1∶50 (Cell Signaling Technology). Secondary antibodies: goat anti-rabbit IgG (1∶400), rabbit anti-goat IgG (1∶400), and rabbit anti-mouse IgG (1∶400). DAPI (1∶1000) marked nuclei.

Hispidulin was purchased from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) (0.1%). Bax, cleaved-caspase-3, cleaved-caspase-9, LC3B-I, LC3B-II, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DAPI, DMSO, Dulbecco's modified Eagle medium (DMEM), and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit and terminal deoxynucleotidyl transferase-meditated dUTP-fluorescein nick end labeling (TUNEL) in situ cell death detection kit were from Roche Diagnostics (Indianapolis, IN, USA). The bicinchoninic acid (BCA) protein assay kit was from Thermo Fisher Scientific, Waltham, MA, USA.

All reagents and chemicals used were kindly offered by Hematopoietic Stem Cell Transplantation Center Laboratory of Guizhou Medical University unless noted otherwise. Fenretinide was purchased from MedChem Express, dissolved in DMSO and stored at -80℃. Leptomycin B was purchased from Solarbio Science&Technology Co.(Beijing,China). The antibodies against cleaved caspase 3, cleaved caspase 9, cleaved PARP and Bax were obtained from Santa Cruz Biotechnology (CA, USA). The antibodies against β-actin, mouse or rabbit IgG were purchased from Beyotime Biotechnology Co. (Shanghai, China). The antibodies against NR4A1 and Cytochrome C (cyt-c) were purchased from MDL biotech Co.(Beijing, China).

Acrylamide, AO, bovine serum albumin (BSA), bromophenol blue, CQ, 4,6-diamidino-2-phenylindol (DAPI), DMBA, ethidium bromide, JC-1 iodide, 3-methyladenine (3-MA), 2-mercaptoethanol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulphate (SDS), N,N,N’,N’-tetramethylene diamine (TEMED) and Trizol were acquired from Sigma Chemical Company, St. Louis, MO, USA. Power SYBR^® Green PCR master mix was obtained from Applied Biosystems, California, USA. Antibodies for Akt, β-actin, β-catenin, cleaved caspase-3, cleaved caspase-9, cytochrome c, GSK-3β, p-GSK-3β^Ser9, p-GSK-3β^Tyr216, PI3K, and Gapdh were purchased from Santa Cruz Biotechnology, USA. Antibodies for ATG5, Bax, Bcl-2, Beclin-1, Histone H2B, LC-3, p-Akt^Ser473, p-β-catenin^{Ser33,Ser37,Thr41}, and p-β-catenin^Ser552 as well as ELISA kits were from Cell Signaling Technology, USA. Alexafluor-488 conjugated anti-rabbit antibody was obtained from Molecular Probes, Inc. (Eugene, OR, USA). Annexin V-FITC, propidium iodide (PI) kit and p62 antibody were purchased from BD Biosciences (San Diego, CA). Nimbolide was obtained from M/s Asthagiri Herbal Research Foundation, Chennai, India. FuGENE transfection reagent was procured from Promega. Oligonucleotide primers were purchased from Sigma Genosys, San Ramon, USA. All other reagents used were of analytical grade.

Cariporide, curcumin, and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against NHE1, MCT1, MCT4, ATP5A, v-ATPase, β-actin, α-tubulin, Rag-A, Rag-B, Raptor, Caspase 3, Caspase 9, cleaved Caspase 3, and cleaved Caspase 9 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against AMPK, p-AMPK, GβL, PRAS40, and p-4EBP1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

A Bio-Rad protein assay system (Bio-Rad Laboratories, Inc.) was used to determine the protein concentrations. The samples (35 μg per lane) were separated by 10–12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer apparatus. The bands were cut into streams by molecular weight, according to the rainbow markers. The protocols for blocking and incubating with primary and secondary antibodies were the same as previously published [50 (link),51 (link),52 (link)]. Primary antibodies including phospho-AKT(S473) (sc-293125, 1:1000), Bcl2 (sc-7382, 1:2000), Bax (sc-7480, 1:500), SOD (sc-101523, 1:1000), CAT (sc-271358, 1:2000), GPx (sc-133160, 1:1000), cleaved caspase-3 (sc-56052, 1:1000), cleaved caspase-9 (sc-56073, 1:2000), β-actin (sc-47778, 1:1000), and horseradish peroxidase (HRP)-conjugated secondary antibodies (sc-2031, 1:15000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-PI3K (4228, 1:2000), and cleaved caspase-7 (9491, 1:2000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Finally, the bands were visualized with enhanced chemiluminescence (ECL) and the ImageJ 1.52v program was used for the quantitative analysis of the intensity of the band signal.