Mouse tnf α

Alisol F and 25-anhydroalisol F were isolated from A. orientale by one of the authors (Q. Ma) using our previously-established method and provided with a purity of 98.0% determined by high-pressure liquid chromatography. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin, and penicillin-streptomycin-amphotericin (PSA) were purchased from Gibco BRL Co. Ltd. (Gaithersbug, MD, USA). Lipopolysaccharide (LPS) from Escherichia coli 055:B5, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dexamethasone (DXM), d-galactosamine (d-gal) and Trizol reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The Griess reagent, the protein extraction kit and BCA protein assay kit were obtained from Beyotime Institute of Biotechnology (Beijing, China). Go Tag^® qPCR Master Mix and GoScript^TM Reverse Transcription System were purchased from Promega (Madison, WI, USA). Rabbit polyclonal antibodies against inducible nitric oxide synthase (iNOS), COX-2, p-p38 (Thr180/Tyr182), p38, p-ERK1/2 (Thr202/Try204), ERK1/2, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, p-p65 (Ser536), p65, GAPDH, p-IκB-α (Ser32), IκB-α, p-STAT3 (Tyr705), STAT3, goat-IgG HRP, and lamin B1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse TNF-α, IL-1β, and IL-6 ELISA kit were from R and D systems (Abingdon, UK).

The antibodies used for immunoblotting included: mouse monoclonal antibody against GAPDH (RM2002, Beijing Ray, Beijing, China); rabbit monoclonal antibodies against HtrA2 (ab75982, Abcam, Cambridge, MA, USA), p-RIPK1 (65746, Danvers, MA, CST, USA), RIPK1 (3493, CST), p-RIPK3 (93654, CST), human p-MLKL (91689, CST), human MLKL (ab184718, Abcam), and mouse p-MLKL (ab196436, Abcam); rabbit polyclonal antibodies against RIPK3 (ab56164, Abcam) and mouse MLKL (ab172868, Abcam); and goat anti-mouse (R3001, Beijing Ray) or goat anti-rabbit (R3002, Beijing Ray) HRP-conjugated secondary antibody.
The antibodies used for IHC staining included: CD11b (ab133357, Abcam), S100a9 (73425, CST, USA), HtrA2 (ab75982, Abcam), MPO (ab9535, Abcam), and F4/80 (ab111101, Abcam).
Other reagents included: DSS (36,000–50,000 kD, MP Biomedicals, Santa Ana, CA, USA), UCF-101 (Cayman Chemical, USA), Nec-1s, BV-6, Z-VAD (Selleck, Houston, TX, USA), mouse TNF-α (R&D, Minneapolis, MN, USA), Cell Counting Kit-8 (CCK-8, MedChemExpress, Monmouth Junction, NJ, USA), FITC-dextran (4 kDa, Sigma, St. Louis, MO, USA).

Total RNA from fresh or cytokine-treated whole islets was isolated using RNeasy Plus Mini Kit (Qiagen). cDNA was prepared with random primers using SuperScript III reverse transcriptase (Life Technologies). For cytokine treatment, isolated islets were recovered overnight in islet media (DMEM containing 1gr/l glucose, 10% vol/vol FBS, 0.1% vol/vol Penicillin/Streptomycin), followed by 24-hr incubation with 10 ng/ml of either mouse IL-1β, mouse TNFα or mouse INFγ (R&D Systems), with the addition of Alk5 inhibitor II (1 µM, Axxora) or vehicle (DMSO) at the same dilution. Relative expression of Ucn3, Ins1, Nkx6.1, Pdx1, and FoxO1 was determined using gene-specific TaqMan probes with TaqMan Fast Universal PCR Master Mix (Life Technologies) on an ABI 7900 Real-Time PCR machine. Relative expression of mouse MafA was determined using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent) on the same machine. Primers for mouse MafA were 5′-AGCGGCACATTCTGGAGAG-3′ forward and 5′-TTGTACAGGTCCCGCTCCTT-3′ reverse. Levels of gene expression were normalized to the expression of Ubc or Eif2A genes.