Nifuroxazide

Recombinant murine IFN-γ and TNF-α were purchased from PeproTech (Rocky Hill, USA). Antibodies against β-actin, iNOS, NF-κB p65, phospho-NF-κB p65, IκBα, histone H3, STAT1, phospho-STAT1 (Tyr701), AKT, phospho-AKT (Ser473), phospho-AKT (Thr308), LAMP1, PPCA, and LAMP-2s (ABL93) were purchased from Cell Signaling Technology (Danvers, USA). Antibodies against LAMP-2A, HSPA8, NFAT1, and IKKα+β were purchased from Abcam (San Francisco, USA). L-NMMA, Fludarabine, MK2206, PDTC, Nifuroxazide, and LY294002 were purchased from Selleckchem (Houston, USA). BAY11-7082 was purchased from MedChemExpress (Shanghai, China). ConA, Griess reagents, and the lysosome isolation kit were from Sigma (St Louis, USA). C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and maintained under specific pathogen-free conditions in the vivarium of the Shanghai Institute of Nutrition and Health of the Chinese Academy of Sciences. All animal experiments were performed according to the guidance of the Institutional Animal Care and Use Committee of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, and complied with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

The following reagents were used: MMC, nifuroxazide, U0126, SP600125, and cycloheximide were purchased from Selleck Chemicals (Houston, TX, USA). Stock solutions of these reagents were prepared in dimethyl sulfoxide (DMSO) or water at a concentration of 5 ~10 mmol/L and they were stored at −20 °C before use. Working solutions of these reagents were diluted from the stock with cell culture medium and they were used at concentrations ranging from 0.5 μmol/L to 20 μmol/L for the treatment of cell lines. The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): MHC-I, p65, phospho-p65, phospho-STAT3, STAT3, c-JUN, phospho-c-JUN, ERK, phospho-ERK, JNK, and phospho-JNK. Human B7H1/PD-L1 polyclonal was purchased from Santa Cruz Biotechnology (Dallas, TX, USA); and IFNGR1 and GAPDH were from Abcam (Cambridge, UK). Polyclonal goat anti-mouse and goat anti-rabbit secondary antibodies were obtained from R&D Systems (Minneapolis, MN, USA).

The third-instar larvae were injected with the Toll inhibitor (BAY 11-7082), Imd inhibitor (parthenolide), JAK/STAT inhibitor mix (20 μM tyrphostin AG 490, Selleck Chemicals, S1143; 5 μM nifuroxazide, Selleck Chemicals, S4182), heat-inactivated B. bassiana conidial suspension, and 0.15 mol/L NaCl solution (as the control), with a volume of 1 μL. A total of 60 larvae were injected in each treatment and performed in triplicate. After 24 h, the injected S. frugiperda larvae were submerged in B. bassiana conidial suspension (10⁸ spores/mL, with 1% penicillin–streptomycin) for 5 s and then reared with fresh corn at 70% humidity and 26°C ± 2°C. The number of dead larvae was recorded every 12 h. A lethal time of 50% (LT₅₀) was used to show the resistance of S. frugiperda larvae against B. bassiana. LT₅₀ and confidence intervals of 95% of each treatment were analyzed and calculated using SPSS21.0.

MSCs were cultured in 12-well or 48-well plates, upon reaching 80–90% confluency, cells were washed with PBS and then treated with indicated stimulations. The stimulations were TNFα (eBioscience, MA, USA) (10 ng/ml), IFNγ (eBioscience, MA, USA) (10 ng/ml), dexamethasone (0.1, 1, 5 or 10 ng/ml) (Sigma, MA, USA), DMSO (Sigma, MA, USA), RU486 (2 μM) (Selleck, WA, USA), BAY 11-7082 (2 μM) (Selleck, WA, USA), nifuroxazide (40 μM) (Selleck, WA, USA) alone or a combination of these two cytokines and inhibitors for indicated times. (n = 3 or 4 in each group)