Fig 2: The novel role of ATF5 in the HIF1 signaling pathway. a Detection of the expression level of identified proteins in shATF5 versus shnc cells by western blot in hypoxia. b Detection of the expression level of identified mRNAs in shATF5 versus shnc cells by RT-PCR; p < 0.01 in hypoxia. c Evaluating the secretion of VEGFA in the identified cells by ELISA; p < 0.01 in hypoxia. d Dual-luciferase assays were performed to detect the luciferase activation of identified genes in ESCA cells transfected with shnc and shKDM4C; p < 0.01 in hypoxia. e Investigating the tube formation ability of HUVECs induced by supernatant from medium of identified cells in hypoxia; Scale bar 100 μm; p < 0.01

Fig 3: Reduction of HIF1α accumulation in UCHL1−/− cells(A) Immunoblot analysis of lysates prepared from HSCs isolated from matched littermate wild-type and uchl1−/− mice. (B) Quantitative RT-PCR analysis of Hif1a mRNA levels in from HSCs isolated from matched littermate wild-type and uchl1−/− mice (all relative expression compared with RPL13A mRNA). Quantitative RT-PCR was performed using four biological replicates. Statistical analysis was performed using GraphPad Prism by a unpaired Student’s t-test; ns>0.05 . (C) HSCs isolated from matched littermate wild-type and uchl1−/− mice exposed to 1% O2 for the indicated times. Whole-cell lysates (WCLs) prepared from these cells were subjected to immunoblot analysis to assess expression levels of the indicated proteins. (D) HSCs prepared from wild-type mice were pre-treated with LDN 57444 (uM) for 30 min before being exposed to 1% O2 for the indicated times. Whole-cell lysates (WCLs) prepared from these cells were subjected to immunoblot analysis to assess expression levels of the indicated proteins. Images presented are representative of three biological repeats.

Fig 4: miR-199a direct targets 3′-UTR region of HIF-1α(A) Target prediction from www.targetscan.org shows the position 31–37 of HIF-1A 3′-UTR contains putative binding sites for miR-199a. (B) Under hypoxia, SaoS-2 (upper) and MG63 (lower) cells were transfected with control miRNAs or miR-199a mimics for 48 h. Cell lysates were prepared for Western blotting analysis. β-actin is a loading control. (C) SaoS-2 (left) and MG63 (right) cells were cotransfected with luciferase reporter plasmids with wild-type 3′-UTR of HIF-1A or mutant 3′-UTR of HIF-1A and miR-199a mimics or control miRNAs using Lipofectamine 2000 reagent. Forty-eight hours post transfection, cells were harvested and lysed with passive lysis buffer. Luciferase activities were measured by a dual luciferase reporter assay. The results were expressed as relative luciferase activity (firefly LUC/Renilla LUC). Columns, mean of three independent experiments; bars, S.E.M. **, P<0.01 and P<0.05 is considered to be of statistical significance.

Fig 5: (a) Treatment of DRG cultures with metformin (20 mM) reduced the levels of HIF1A, activated AMPK which is confirmed by increased phosphorylation of Thr172 and suppressed translation which is validated by reduction in the phosphorylation of rS6 (HIF1A, *P= 0.0119; AMPK, **P= 0.0046; rS6, *P= 0.0148; six wells/group). (b) Co-injection of metformin (Met, 150 mg/kg) with bortezomib (Bort) for five consecutive days prevented the development of bortezomib-induced neuropathic pain (****P<0.0001, five mice/group). HIF1A: hypoxia-inducible factor 1 alpha; AMPK: AMP-activated protein kinase.