Akt inhibitor 8

MDMs (5 × 10⁶ cells) were treated with recombinant myristoylated Nef protein (rNef) from the SF2 HIV-1 strain (1–100 ng/mL) (cat # PR-382, Jena Bioscience, Jena, Germany). Cell pellets were collected at various times after rNef treatment, washed extensively, and either lysed before Western blot analysis or fixed with BD Cytofix (BD Biosciences, San Jose, CA, USA) for 20 min before flow cytometric analysis. MDMs were treated with the Akt inhibitor VIII (cat # sc-202048A) (0, 25, 50 µM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h before addition of rNef (100 ng/mL) for 30 min.

PBLs (5 × 10⁶ cells) were treated with recombinant myristoylated Nef protein (rNef) from the SF2 HIV-1 strain (100 ng/ml) (cat # PR-382, Jena Bioscience, Jena, Germany). Cell pellets were collected at various times after rNef treatment, washed extensively and either lysed before western blot analysis or fixed with BD Cytofix (BD Biosciences, San Jose, CA) for 20 min before flow cytometric analysis. Some PBLs were treated with the PI3K inhibitors LY294002 (0, 25 μM, 50 μM) or Wortmannin (0, 0.5 μM, 1 μM) (Cell Signaling Technology, Beverly, MA), or with the Akt inhibitor VIII (cat # sc-202048A) (0, 25, 50 μM) (Santa Cruz Biotechnology, Santa Cruz, CA) for two hours before addition of rNef (100 ng/ml) for 30 min.

After isolation, hepatocytes were plated onto 60-mm mouse collagen IV-coated dishes (BioCoat, Becton–Dickinson) at a density of 1 × 10⁶ viable cells/dish in DMEM medium containing 10 % FBS [44 (link)]. They were allowed to attach overnight prior to experiment. After overnight serum deprivation, hepatocytes isolated from wide type mice were stimulated with 100 nM insulin or PBS for 10 min and Akt phosphorylation was assessed by western blot. To further confirm whether insulin could induce Akt phosphorylation in diabetic hepatocytes, after overnight serum deprivation, cells were incubated with or without 1 μM pAkt inhibitor VIII for 4 h and then stimulated with 100 nM insulin for 10 min.
To evaluate the effect of insulin on ANGPTL3 production, hepatocytes were serum starved for 24 h and then exposed to 100 nM insulin in the presence of absence of 1 μM Akt inhibitor VIII (Santa Cruz, CA, USA) for another 24 h. Cells were collected for western blot to study ANGPTL3 expression. In parallel, HepG2 were maintained in the same culture medium. After they reached 90 % confluency, they were serum starved and treated with 100 nM insulin as murine hepatocytes.