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. 2020 Jul 23;10(20):9230–9248. doi: 10.7150/thno.45253

Figure 6.

Figure 6

Downregulation of miR-17 impaired glucose uptake by promoting oxidative stress via targeting keap1. A-D. The representative western blot images (A) and quantification (B-D) of genes related to oxidative stress. Gapdh was used as the loading control. C2C12 myotubes were transfected with 50 nM agomiR-17 or 200 nM antagomir-17 for 48 h (n = 3 independent experiments). E. The miR-17 target regions in 3'UTR of Keap1. Mmu, mouse; Rno, rat; Hsa, human. WT: a truncated Keap1-3'UTR with wild-type miR-17 binding site; MT: a truncated Keap1-3'UTR with mutated miR-17 binding site. F-G. The relative luciferase activity in C2C12 myoblasts transfected with reporter vector containing the wild-type (F) or mutated (G) Keap1 3'UTR together with agomiR-17, antagomiR-17 or corresponding controls (n = 4 independent experiments). H-J. The representative western blot images (H) and quantification (I-J) of pAkt and mGlut4 in C2C12 myotubes treated with antagomiR-17 (n = 3 independent experiments). Gapdh was used as the loading control. K. The insulin-stimulated 2-DG uptake in C2C12 myotubes treated with antagomiR-17 (n = 3 independent experiments). L. The expression of miR-200a in C2C12 myotubes transfected with agomiR-17 and antagomiR-17 (n = 3 independent experiments). U6 was used to normalized miR-200a expression. M-P. C2C12 myotubes were transfected with 50 nM agomiR-17 and treated with 25 µM DEHP (n = 3 independent experiments). M-N. The mRNA expression of Keap1 and Nrf2. Gapdh was used as the loading control. O. The calculated GSH/GSSG ratio. The levels of GSH and GSSG were shown in Figure S8C-D. P. The insulin-stimulated 2-DG uptake. Q-S. C2C12 myotubes were co-treated with 25 µM DEHP, antagomir-17, SFN or corresponding controls (n = 3 independent experiments). Q. The expression of miR-200a normalized by U6. R. The mRNA expression of Txnip. Gapdh was used as the loading control. S. The insulin-stimulated 2-DG uptake. All data were presented as the mean ± SEM. *P < 0.05, **P < 0.01 vs. corresponding control as indicated.