D glucose g8270

LPF peptide was synthesized by Genscript Biotech Corporation (Nanjing, China). D-glucose (G8270) and MTT (M2003) were purchased from Sigma. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH, A101386) was from Aladdin Biochemical Technology (Shanghai, China). Sodium fluorescein and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were from Wako Pure Chemical (Osaka, Japan). Anti-O-GlcNAc antibody (CTD110.6) (sc-59623) was from Santa Cruz Biotechnology. Lipid Peroxidation Assay Kit (S0131S), Glutathione (GSH) Assay Kit (S0052), Hematoxylin and Eosin Staining Kit (C0105S), DAPI (C1002), and H₂DCFDA (S0033S) were purchased from Beyotime Biotechnology (Shanghai, China). Anti-Pax3 antibody was from DSHB. Primary antibodies, anti-NRF2 and anti-Keap1, were purchased from Proteintech. Anti-β-Actin and HRP-conjugated secondary anti-rabbit and anti-mouse antibodies were purchased from Fude Biological Technology (Hangzhou, China). Alexa Fluor 488 goat anti-rabbit IgG was purchased from Invitrogen (Carlsbad, CA, USA).

Glucose tolerance was assessed in mice using an IPGTT, which was performed by injecting 1.5 g/kg D-glucose (G8270, Sigma-Aldrich, MO, USA) intraperitoneally into overnight-fasted mice. Tail blood was collected before and 0, 15, 30, 60, 90, and 120 min after glucose administration. Blood glucose concentrations were measured using an Accu-Check glucose analyzer (Roche Diagnostics Ltd., Mannheim, Germany). Body weights, fasting glucose levels, and area under the IPGTT curve were analyzed using two-sample t-test. Data are presented as means ± standard errors of means, and statistical significance was accepted for P values <0.05.

Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and trypsin were purchased from Gibco Life Technologies (Grand Island, NY, USA). D-Glucose (G8270) and N-acetyl cysteine (NAC; A7250) were purchased from Sigma Chemical (St. Louis, MO, USA). The JNK inhibitor SP600125 was purchased from Selleck Chemicals (s1460; Houston, TX, USA). The antibodies used in this study were against CDK2 (ab32147), active caspase-3 (ab2302), and Ki-67 (ab16667; Abcam Inc., MA, USA) and GAPDH (5174S), JNK (9252T), phospho-JNK (4668T), c-Jun (9165P), phospho-c-Jun (3270P), cyclin D1 (2978), p21 (2947), Bax (2772S), and Bcl-2 (15071S; Cell Signaling Technology Inc., MA, USA).

Bone marrow cells were isolated from femurs of 3–6 months old male TG2 null mice and their wild type counterparts. Bone marrow macrophages (BMDMs) were differentiated in DMEM supplemented with 10% FBS (12106C), 2 mM L-glutamine (G7513), 1 mM Na-pyruvate (S8636), 50 μM 2-mercaptoethanol (M3148) and 100 U/ml penicillin/100 μg/ml streptomycin (P4333) all from Sigma-Aldrich and 10% L929 fibroblast conditioned media for 7 days. For metabolic activation, differentiated macrophages were treated with a combination of 30 mM D-glucose (G8270), 10 nM insulin (12643) and 0.4 mM sodium-palmitate (P9767) all from Sigma-Aldrich for 24 h (16). Sodium palmitate was prepared by diluting a 200 mM stock solution in 70% ethanol into 10% fatty acid-free, low-endotoxin BSA (Sigma Aldrich, A8806 adjusted to pH 7.4) to obtain a 5 mM palmitate-BSA stock solution that was filtered using a 0.22-μm low-protein binding filter (Millipore). BSA/ethanol was used in control treatments during the protocol. In some experiments during the 24 h metabolic activation BMDMs were treated with PP2 (Sigma Aldrich, 529573), a reversible ATP-competitive inhibitor of the Src family of protein tyrosine kinases, in 2 µM final concentration or 0.5 mg/ml RGD peptide (Cayman Chemical, 529573).

Streptozocin (S0130) and D‐glucose (G8270) were obtained from Sigma‐Aldrich Chemical Company (Sigma). BBR (BWC9020‐2016) was procured from BeNa Biotechnology. Membrane and cytosolic protein extraction kits (P0033), trypan blue (ST798), EdU (5‐ethynyl‐2′‐deoxyuridine) assays (C0071S) and cell cycle detection kits (BB‐4104) were acquired from Beyotime Biotechnology. The 2‐NBDG (2‐deoxy‐2‐ [(7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl) amino]‐D‐glucose) assay (N13195) was obtained from Thermo Fisher Scientific. Anti‐PI3K‐p85 (#4292), anti‐Akt (4691S), anti‐p‐Akt (#4060), anti‐AS160 (#2670), anti‐p‐AS160 (#8619) and anti‐GAPDH (#5174) antibodies were obtained from Cell Signaling Biotechnology. Rabbit anti‐GLUT1 (ab115730) and anti‐Cyclin D1 (ab16663) antibodies were purchased from Abcam Biotechnology (Abcam).

Hispidulin (1447-88-7) was purchased from Victory Biological Technology, China and D-glucose (G8270) was from Sigma, USA.