Fig. 3
CAMK4 overexpression triggered autophagy and inhibited IR in HTR-8/SVneo cells under IR condition. HTR-8/SVneo cells were transfected with CAMK4 overexpression plasmid or vector for 24 h, followed by incubation with or without 10–6 mol/L insulin for 48 h (A–C). A Representative western blot images and relative protein levels of Beclin 1, p62, LC3 I and LC3 II in HTR-8/SVneo cells were displayed. B HTR-8/SVneo cells were infected with adenovirus vector carrying mRFP-GFP-LC3, and the red/green fluorescence was observed. Scale bar = 50 μm. C The levels of LC3 I/II in HTR-8/SVneo cells with 10–6 mol/L insulin pre-treatment, CAMK4 overexpression or/and CQ administration. HTR-8/SVneo cells were pre-incubated with or without 10–6 mol/L insulin for 48 h, followed by stimulation with 10–7 mol/L insulin for 15 min, and the transfection of CAMK4 overexpression plasmid or empty vector was performed 24 h before insulin pre-incubation (D–H). D Glucose uptake ability was assessed in cells of each group. E–H Theratio of Phospho.IRS-1Ser307/Total.IRS-1 (E), Phospho.IRS-1Tyr612/Total.IRS-1 (F) or Phospho.AKTSer473/Total.AKT (G) in HTR-8/SVneo cells wasdetected by western blot. Representative western blot images of phosphorylated IRS-1 at Ser307, phosphorylated IRS-1 at Tyr612, total IRS-1, phosphorylated AKT at Ser473, total AKT were displayed (H). I Dual-luciferase reporter assay was performed to assess the effect of CAMK4 overexpression on luciferase activity of NUR77 in HTR-8/SVneo cells. J and K HTR-8/SVneo cells were pre-incubated with or without 10−6 mol/L insulin for 48 h, and the transfection of CAMK4 overexpression plasmid or empty vector was performed 24 h before insulin pre-incubation. The mRNA and protein levels of NUR77 in HTR-8/SVneo cells were detected by real-time PCR (J) and western blot (K), respectively. Error bars depict the standard deviation of the mean. IR, insulin resistance; −, no treatment; CQ, chloroquine; *p < 0.05, ***p < 0.001