Jnk1 2

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Escherichia coli lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Interleukin-1 beta (IL-1β), IL-6, IL-10, and tumor necrosis factor alpha (TNF-α) Elisa kits were obtained from eBioscience (Science Center Drive San Diego, CA, USA). COX-2 Elisa kit was purchased from Axygen (Central Avenue Union City, CA, USA). Antibodies against IκBα, p-IκBα, NF-κB p65, p-NF-κB p65, p38α, p-p38α, JNK1/2, p-JNK1/2, ERK1/2, p-ERK1/2, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and secondary antibodies were obtained from Sangon Biotech (Shanghai, China). Other chemicals and reagents were all analytically pure and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

PTER of 98% purity was purchased from Enzo Life Sciences (Lausen, Switzerland). A 100 mM stock solution of PTER was made in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) and stored at −20°C. The final concentration of DMSO for all treatments was <0.5%. Antibodies, specifically of cleaved caspase-3, caspase-8, caspase-9, poly(ADP-ribose) polymerase (PARP), heat shock protein 70 (HSP70), p-extracellular signal-regulated kinase (ERK)1/2, p-p38, p-c-Jun N-terminal kinase (JNK), ERK1/2, p38, JNK1/2, CDK 2, cathepsin B, C23, and β-actin (for the Western blot analysis), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against cyclin-dependent kinase (CDK)6, p21 Cip1, p27 Kip1, p15 INK4B, and cyclin D3 were purchased from Cell Signaling Technology (Danvers, MA). Anti-cyclin A2 and anti-cyclin E2 antibodies were purchased from Epitomic (Burlingame, CA). 4′-6-Diamidino-2-phenylindole (DAPI) was purchased from Sigma. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, the ERK1/2 inhibitor, U0126, and the JNK1/2 inhibitors, SP600125 and JNK-IN-8, were purchased from Calbiochem (San Diego, CA). The caspase-3 inhibitor, Z-DEVE-FMK, was purchased from BioVision (Mountain View, CA). Unless otherwise specified, other chemicals used in this study were purchased from Sigma.

Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin, and penicillin were obtained from Life Technologies (Carlsbad, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IFN-γ, and IL-2 were from R&D Systems (Minneapolis, MN, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from USB Corp. (Cleveland, OH, USA), and the LDH release detection kit was obtained from Roche Applied Science (Indianapolis, IN, USA). All kits were used according to the manufacturers’ protocols. CTX monohydrate, LPS, concanavalin A (ConA), and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). YAC-1 cells, a mouse lymphoma cell line, and RAW264.7 cells, a mouse macrophage cell line, were obtained from ATCC (Manassas, VA, USA; nos. ATCC^®TIB-160^TM and ATCC^®TIB-71 ^TM). Antibodies against β-actin, iNOS, p-NF-κB p65, NF-κB p65, p-IκB, IκB, p-Akt, Akt, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-p38, and p38, and HRP-linked anti-rabbit IgG secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were of the highest grade commercially available.

After being subjected to the indicated treatments, approximate 5 × 10⁶ BM-MSCs were washed with cold PBS three times and lysed with cell lysis solution. Sample buffer was added to cytosolic extracts, and after boiling for 10 min, equal amounts of supernatant from each sample were fractionated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature. Then the membranes were incubated with antibody for p-PERK, PERK, p-IRE1, IRE1, BAX, Bcl-2 (1 : 800; Cell Signaling), caspase-3, eIF 2α, CHOP, p-JNK1/2, JNK1/2, p-extracellular signal-regulated kinase (ERK), ERK, p-p38, p38 (1 : 800; Santa Cruz) at 4°C overnight. At last, immunoreactive bands were then detected by incubation with conjugates of anti-rabbit IgG with horseradish peroxidase (1 : 2,000; Southern-Biotech) for 2 h and visualized by using enhanced chemiluminescence system (ECL; Pierce Company, USA).

BM-MSCs grown on coverslips were fixed with 4% paraformaldehyde at 4°C for 40 min, followed by several rinses in phosphate-buffered saline (PBS). Nonspecific binding sites were blocked at room temperature for 2 h with 5% normal horse serum (Sigma-Aldrich, St. Louis, MO, USA) or normal donkey serum (Sigma-Aldrich, St. Louis, MO, USA) diluted in 0.1% Triton X-100-PBS. BM-MSCs were then incubated overnight at 4°C with primary antibodies: CHOP (Cell signaling, MA, USA), JNK1/2 and caspase-3 (green; Santa Cruz, CA, USA) at 1 : 300 dilution with blocking buffer, followed by a mixture of fluorescein isothiocyanate and tetramethylrhodamineisothiocyanate-conjugated secondary antibodies (BD Biosciences) for 2 h at 4°C. BM-MSCs were finally incubated with DAPI (blue; Sigma, MO, USA), which was used to stain the nucleus for 30 min at 37°C. The slides were visualized by using a fluorescent microscope (Leica, Microsystems, Germany). All photomicrographs shown in this study were representative of multiple experiments.