Gsk2606414

According to the previous description, murine MPCs were collected from the hind limb muscle of neonatal B6 mice (Sciorati et al., 2015 (link)). MPCs were cultured in Dulbecco’s modified Eagle’s medium Nutrient/F12 (DMEM/F12, Hyclone, United States) containing with 10% fetal bovine serum (FBS, Gibco, United States), 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate in a 5% CO₂-humidified chamber (Heraeus, Germany) at 37°C. For differentiation studies, MPCs were cultured in differential medium (DM) (DMEM, added with 2% horse serum) for 72 h. For pro-inflammatory stimuli, MPCs were treated with IFN-γ (60 ng/mL, R&D, United States) for 24 h (Nozaki et al., 2010 (link)). For inhibiting UPR and the branches, the cells were co-cultured with 4-PBA (10 mM, Selleck, China), 4μ8c (50 μM, Selleck, China), and GSK2606414 (1 mM, Selleck, China), respectively, under pro-inflammatory stimuli. TM (1 μg/mL, Santa Cruz, United States), or TG (0.2 mmol/L, Santa Cruz, United States) were used for 4 h as positive control for testing UPR activation and as UPR activators. Cells were cultured in DM with or without 20 mM SB202190 (Sigma, United States) to blocking p38 activation in IFN-γ medium.

Antibodies against tubulin (mouse polyclonal, 1:5000), actin (mouse polyclonal, 1:5000), PERK (bovine monoclonal, 1:500, sc-9481), p-PERK (rabbit polyclonal, 1:1000, sc-32577), cyclin D1 (mouse polyclonal, 1:500, sc-20044), and HPV E7 (rabbit polyclonal, 1:1000) were from Santa Cruz. Antibodies against Eif2a (rabbit polyclonal, 1:1000, 5324), p-Eif2a (rabbit polyclonal, 1:1000, 9721), Phospho-Akt (Thr308) (1:1000, 13038), Phospho-NF-κB p65 (Ser536) (1:1000, 3033), ATF4 (rabbit polyclonal, 1:1000, 11815), CHOP (rabbit polyclonal, 1:1000, 5554), cleaved-caspase 3 (rabbit polyclonal, 1:1000, 9661), and cleaved-PARP (rabbit polyclonal, 1:1000, 5625) were from Cell Signaling Technology. Anti-Ki67 (rabbit polyclonal, 1:2000, Ab15580) antibody were purchased from Abcam. Anti-LMP1 (mouse monoclonal, 1:200, M0897) antibody was from DAKO. Anti-Myc (rabbit polyclonal, 1:1000, PAB12716) antibody was from Abnova. Anti-FLAG (mouse monoclonal, M2, 1:1000, F3165) antibody was from Sigma-Aldrich. The PERK inhibitor (GSK2606414) was purchased from Selleck. Other chemicals, as well as Endoplasmic Reticulum isolation kit (ER0100) were purchased from Sigma-Aldrich.

Stock solutions of reagents were prepared by dissolving the reagents in dimethyl sulfoxide (DMSO, Sigma-Aldrich). SBI-0206965, GSK 2606414, and MRT 68921 were purchased from Selleck Chemicals (Houston, TX, USA). Bafilomycin A1 and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich. Hydroxychloroquine (HCQ) was purchased from Myung-in Pharmaceuticals. z-VAD-FMK was obtained from R&D Systems. Control cells were treated with equal amounts of DMSO.

Male BALB/c mice (8–10 weeks) were purchased from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and raised by the Animal Experiment Center of Shanghai University of Traditional Chinese Medicine. All animal experiments were conducted in accordance with the procedures approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine.
HCT116 and HT29 cells were purchased from Sailybio (Shanghai, China). RPMI 1640, penicillin and streptomycin, fetal bovine serum (FBS), BSA, ER-tracker TM Green and Lipofectamine^TM 2000 Transfection Reagent were purchased from Invitrogen (Carlsbad, CA, United States). AOM, thapsigargin, salubrinal and 4-PBA were obtained from Sigma-Aldrich (St. Louis, MO, United States). PrimeScript^TM RT Master Mix, TB Green Premix Ex Taq^TM and RNAiso Pius were purchased from TaKaRa (Tokyo, Japan). GSK2606414 and Alisertib were purchased from Selleckchem (Houston, TX, United States). DSS was purchased from MP Biomedicals (Santa Ana, CA, United States). PVDF membranes were purchased from Millipore (Billerica, MA, United States).

Sorafenib was purchased from Selleck Chemicals and dissolved in DMSO as a 10 mM stock solution, aliquoted and stored at −80°C. Sorafenib treatment was performed when cells had reached ~ 80% confluence. Before treatment, cells were cultured in DMEM supplemented with 5% FBS. PERK inhibitors, GSK2606414 and GSK2656157, were purchased from Selleck Chemicals and EMD Millipore, respectively. Both PERK inhibitors were dissolved in DMSO as a 10 mM stock solution, aliquoted and stored at −80°C. Cycloheximide and puromycin were purchased from Sigma (St Louis, MO) and dissolved in water as a 10 mg/mL and 25 mg/mL stock solutions, respectively, aliquoted and stored at −20°C.

Cerulein was purchased from MedChem Express (HY-A0190, Shanghai). Lipopolysaccharide (LPS) was purchased from Sigma (L2630, Shanghai). ABT702 was purchased from Merck Millipore (116890, Shanghai). ZM241385 (an adenosine A_2A receptor antagonist) and GSK2606414 (PERK inhibitor) were purchased from Selleck (S8105 and S7307, respectively, Shanghai). The following primary antibodies were purchased: ß-actin antibody (Proteintech, 66009), ADK antibody (Abcam, ab227087), RIP1 antibody (CST, 3,493), phosphor-RIP1 (Ser161) antibody (Affinity, AF7377), RIP3 antibody (CST, 15828), phosphor-RIP3 antibody (Abcam, ab195117), mouse MLKL antibody (Proteintech, 66675), rat MLKL antibody (Abcam, ab243142), phosphor-MLKL antibody (Affinity, AF7420), NF-κB p65 antibody (CST, 8242), phosphor-NF-κB p65 antibody (CST, 3,033), GRP78 Antibody (Proteintech, 11587), CHOP antibody (Proteintech, 15204), PERK antibody (Proteintech, 24390), phosphor-PERK antibody (CST, 3,179), eIF2α antibody (CST, 5324), phosphor-eIF2α antibody (CST, 3,398), CD11b antibody (Abcam, ab133357), MOMA-2 antibody (Abcam, ab33451), and amylase antibody (Santa Cruz Biotechnology, sc-46657). Secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States).

MDA-MB-231, MCF7, ZR-75-1, and HFF cell lines were purchased from the ATCC. ZR-75-1 cells were cultured in RPMI 1640 (Gibco), and the other cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Excell Bio) in an incubator at 37 °C with 5% CO₂. The antibody against GAPDH (sc-47724) was purchased from Senta Cruz Biotechnology. Antibodies against STING (13647), TBK1 (3504s), pTBK1 S172 (5483S), elF2α (5324S), and p-elF2α S51 (3398S) were purchased from Cell Signaling Technology. Antibodies against PLAU (ab24121) and pIRF3 S386 (ab76493) were purchased from Abcam (MA 02453, USA). Antibodies against PERK (A18196) and p-PERK T982 (AP0086) were purchased from ABclonal Technology. STING agonist diABZI (Compound 3) and PERK inhibitors GSK2656157 and GSK2606414 were purchased from Selleck Chemicals.