2 n 7 nitrobenz 2 oxa 1 3 diazol 4 yl amino 2 deoxyglucose 2 nbdg

Antibody against pYAP127 (D9W2I), YAP (D8H1X) was purchased from Cell Signaling Technology. The secondary fluorescent antibodies and H&E staining kit were from Abcam. Glut1 inhibitor Bay‐876 was obtained from MCE Med Chem Express. The fluorescent glucose analog 2‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl) amino)‐2‐deoxyglucose (2‐NBDG) was from Cayman Chemical Company. TEAD1 siRNAs were purchased from Santa Cruz Biotechnology. TRAP staining kit was purchased from Sigma. Plasmids pRL‐TK, pcDNA3.1, pcDNA3.1‐flag‐YAP, and shYAP1/2 were obtained from Addgene. The transfection was performed as we previously reported.¹, ⁵⁴, ⁵⁵

SPC (>98% purity), EPL (phosphatidylcholine 84.5%, phosphatidyl-ethanolamine 9%, sphingomyelin 3%, lysophosphatidylcholine 3%, lysophosphatidyl-ethanolamine 0.5%) were kindly provided by Doosan Corporation (Seoul, Korea). 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Chrysin and the solvents used in this study were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Taqman® Universal master mix, Taqman^® probes (5′-fluorescein based reporter dye; 3′-TAMRA quencher) and the high capacity RNA-to-cDNA kit were purchased from Applied Biosystems (Foster City, CA, USA).

HepIR mouse liver cells were cultured in MEMα (Gibco) culture medium supplemented with 4% fetal bovine serum (FBS; Gibco) 100 U/ml penicillin and 100 U/ml streptomycin (Gibco) at 37°C. Cells were pre-incubated in serum-free medium for 4 h and then treated with 200 uM PA to induce insulin resistance.
The glucose uptake of HepIR cells was evaluated using the fluorescent glucose 2-[N-(7-nitrobenz-2-oxa-1,3diazol-4-yl) amino]-2-deoxyglucose (2-NBDG) (Cayman Chemical, Ann Arbor, MI, United States). The effect of GvAEx on glucose uptake was assessed in PA induced insulin-resistant HepIR cells. Briefly, PA induced insulin-resistant HepIR cells were treated with GvAEx (0.1–2 mg/ml) or vehicle in glucose-free DMEM for 1 h. After aspirating the supernatant, the cells were incubated with insulin (10 nM) (Solarbio, Beijing, China) or saline for 10 min. The cells were then gently rinsed with HBSS and incubated with 100 ug/mL 2-NBDG at 37°C for 50 min. The cells were washed with HBSS and collected for further analysis. The fluorescence intensity of the cells was detected by a microplate reader (excitation/emission = 465/540 nm). Relative 2-NBDG (%) uptake was calculated using the following equation (intensity of treatment/intensity of normal control) × 100%.