Fig. 6

CircMYH9 was activated by the ROS-HIF1α pathway under amino acid deprivation. A-B The levels of circMYH9 and MYH9 pre-mRNA were determined by qRT-PCR in CRC cells cultured in complete, SG-free or Glu-free medium with or without NAC treatment. C-D The levels of HIF1α were determined by IB in complete, SG-free or Glu-free medium with or without NAC treatment. E A schematic of the six MYH9 promoter regions (1–6) analysed for HIF1α binding affinity. HBS1 and HBS2 referred to as HIF1α binding sites. F The HIF1α-binding motif enriched in the MYH9 promoter was predicted by JASPAR. G ChIP-qPCR analysis was used to determine the binding affinity of HIF1α to six MYH9 promoter regions in HCT116 cells, showing that p53 bound to HBS1(-1218/-1117) and HBS2(-565/-508) in the MYH9 promoter. ChIP-qPCR with IgG was performed as the control. H-I Luciferase reporter gene constructs were cotransfected with HIF1α shRNA or HIF1α overexpression plasmid in HCT116 cells, and reporter gene activity was measured after 48 h by a dual luciferase assay. The relative value in HCT116 cells cotransfected with shCtrl or vector was set to 100%. Data are shown as the mean ± SD of three independent experiments (*, P < 0.05; **, P < 0.01)