Bms 303141

UT-SCC-5, UT-SCC-8, UT-SCC-9, UT-SCC-11, UT-SCC-15, UT-SCC-19A, UT-SCC-24A, UT-SCC-29, UT-SCC-38, UT-SCC-40, UT-SCC-45, FaDu (Kindly provided by Prof Grenman, University of Turku), UM-SCC-6, UM-SCC-47 (Kindly provided by Dr Carey, University of Pittsburgh), 93-VU-147T (Kindly provided by Dr Dorsman, Amsterdam University Medical Center), and UPCI:SCC-154 (DSMZ) were cultured in DMEM medium (Gibco) supplemented with 4.5 g/L glucose, GlutaMAX, 10% FBS, 100 u/mL penicillin/streptomycin, non-essential amino acids (Gibco), HEPES (Gibco), and sodium pyruvate (Gibco). U2OS DRGFP cells (provided by Prof Tim Humphrey, University of Oxford) were cultured in DMEM supplemented with 10% FCS and 5 mg/mL puromycin and 100 U/mL penicillin/streptomycin. Cells were treated with 5 µM BMS303141 (Sigma-Aldrich, St. Louis, MO, USA) in DMSO or 10 µM C75 (Merck) in DMSO for 6 h prior to irradiation. Single dose irradiation was delivered using a 320 kV XRAD irradiator (RPS Services Limited, Surrey, UK)) at a dose rate of 3.1 Gy/min. An overview of cell line characteristics and tissue origin can be found in Table S1.

The following drugs were used following solubilization in DMSO: tivozanib (Selleck, 43.96 mM stock), cerulenin (TEBU, 50 mM stock), KH-CB19 (R&D Systems, 10 mM stock), bafilomycin (50 μM stock), pimozide (10 mM stock), W7 (53 mM stock), TOFA (10 mM stock), BMS-303141 (25 mM stock) and (15:3)-anacardic acid (10 mM stock; all from Sigma). To determine the step of the viral cycle affected by chemical compounds, HEK-293T cells (seeded the day before on poly-L-lysine-coated plates) were either treated 2 h before or 2 h after cell infection with CHIKV C21 (MOI 40) and left until 8 h p.i. Viral infection was quantified by flow cytometry measurement of the % of capsid-expressing cells. Equal amounts of supernatants (3 μl) from the 2 h post treatment conditions were then measured for viral infectivity on Vero cells (final volume: 150 μl). The percentage of infected cells was measured by flow cytometry at the end of the experiment. For cell viability analysis, we took advantage of the high-precision measurement of the acquired volumes made by the MACSQuant VYB cytometers. Then, knowing the initial volume of cells, the acquired volume and the acquired number of events, we estimated the total number of cells per well. Cell staining for these experiments was performed in V-bottom 96-well plates (Nunc).

Lung DC were isolated from WT mice using the tissue preparation protocol described above. Following the establishment of a single-cell suspension, cells were incubated for 1 h on ice with anti-MHCII beads (Miltenyi) and the enriched fraction was collected using LS MACS columns (Miltenyi). Cells were then stained and cDC1 and cDC2 were sorted. DC were then incubated in RPMI 1640 (without glucose) with 20% fetal calf serum, glutamine, HEPES, penicillin/streptomycin and low (10 mM) or high (50 mM) glucose for 20 h before either analysis or replacement with fresh low-glucose medium and the addition of T cells (see below). Where indicated, either 2-DG (5 mM; Sigma), BMS303141 (10 mM; Sigma) or ANA (20 mM; Sigma) was added.

Immortalized human hepatocytes (IHH) were cultured under standard conditions using control media DMEM/F-12 (Sigma) without phenol red, 10% FBS (Life Technologies, 10,108–165), 1% PEN/STREP (Life Technologies 15,140–122), 0.1% L-Glutamine (Life Technologies 25,030–024), 0.02% dexamethasone (Sigma D49025MG) and 1 pM insulin human recombinant zinc (Life Technologies 12,585–014). For generating steatosis, cells were supplemented with oleic acid-albumin (Sigma O3008) (300 μM). After for 4 days, confluent cells were treated with 200 μM etomoxir (Sigma E1905), 50 μM BMS 303,141 (Sigma SML0784), 5 μM garcinol (Thermo Fisher 15,716,585), acetyl-CoA carboxylase (ACC) inhibitor firsocostat (GS-0976) (Selleckchem S8893) 50 nM, antioxidant supplement (Sigma A1345) 10 × , methotrexate (Sigma M9929) 10 nM and 50 μM ACCSi—CAS 508186–14-9 (Sigma 5,337,560,001) for 4 h at 37 °C.

BMS 303141 (BMS), Tubastatin A (TubA), MG-132 and cycloheximide were obtained from Sigma-Aldrich (SML0784, SML0044, M7449, and C104450, respectively). MG-132 (20 µM) was added to fully differentiated cells 4 h prior to protein extraction. For ubiquitinated Tau MG-132 (17.5 µM) was added to HEK293 transfected cells 15 h prior to immunoprecipitation. Treatment of TubA, (2 µM) for 24 h alone or together with MG-132 (20 µM) for the last 4 h were added to fully differentiated cells, prior to protein extraction. BMS (1 µM) was added to fully differentiated cells for 72 h prior to cells fixation and immunofluorescence analysis.

C57BL/6J male mice were purchased from the Laboratory Animal Center of the Third Military Medical University. Mice were fed a normal chow diet (#1025, fat content 4% by calorie, HFKbio) or high-fat diet (D12492, fat content 60% by calorie, Research Diets) starting at 6 weeks of age for 10–24 weeks. IR-61 or BMS-303141(#SML0784, Sigma) was administered by intraperitoneal injection at 2 mg kg−1 body weight weekly. For the prevention groups, IR-61 was administered concurrently with the NCD or HFD feeding at 6 weeks of age. For the treatment groups, IR-61 was administered by i.p. injection starting at 18 weeks of age (after 12 weeks of HFD feeding). For the IR-61 + Acly inhibitor group, after receiving HFD for 8 weeks, mice were administered the treatment compounds for another 2 weeks. The environmental conditions in the mouse facility were: 12 h light and 12 h dark cycle, temperature range of 21–23 °C, humidity range of 40–50%, and free access to food and water. All animal use was approved by the Ethics Committee and performed in accordance with the Animal Care and Use Committee Guidelines of the Third Military Medical University.