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. 2021 Feb 26;12:1341. doi: 10.1038/s41467-021-21535-3

Fig. 5. HIFAL transcription is activated by HIF-1α transcriptional complex containing HIFAL itself in a feed-forward loop.

Fig. 5

a HIFAL transcription is not in line with HIF-1α protein level after hypoxia. b HIF-1α binds to the HIFAL promoter under hypoxia. MDA-MB-231 cells were cultured under hypoxia for 24 h, then collected for ChIP assay. Primers spanning the HIFAL locus were used to confirm the binding specificity of HIF-1α at the promoter/HRE element. [HIFAL (B)], −916~−719bp [HIFAL (A)], and +578~+749 bp [(HIFAL(C)] of the promoter/HRE element. The β-actin promoter was used as the negative control. c Hypoxia-induced HIFAL transcription is inhibited by HIF-1α knockdown. MDA-MB-231 cells transiently transfected with siHIF-1α were collected to detect the HIFAL promoter transcription activity under hypoxia. d HIF-1α activates HIFAL transcription in a dose-dependent manner under hypoxia.HIF-1α plasmid (0, 0.25, 0.5, 1 μg) was transfected into MCF-7 cells for the detection of HIFAL transcriptional activity. e HRE mutation abolished the hypoxia induced HIFAL transcription activity. MDA-MB-231cells transfected with indicated wild type or mutant HIFAL promoter were collected to detect the promoter transcription activity. f PKM2 binds to the HIFAL promoter under hypoxia. MDA-MB-231 cells were collected for ChIP analyses. g Knocking down PKM2 reduces HIFAL level upon hypoxia, which could not be rescued by PKM1 overexpression. h PKM2 siRNA decreases the hypoxia-induced HIFAL promotor transcriptional activity, which could not be rescued by PKM1 overexpression. i PKM2 activates the HIFAL transcription in a dose-dependent manner under hypoxia. PKM2 plasmid (0, 0.25, 0.5, 1 μg) was transfected into MCF-7 cells for the detection of HIFAL transcriptional activity. j HIFAL binds to HIFAL promoter under hypoxia. MDA-MB-231 cells were transfected with biotinlabeled HIFAL or antisense, then collected for ChIP analyses. k, l Knocking down HIFAL decreases the hypoxia-induced HIFAL promotor transcriptional activity. The transcription activity of HIFAL promotor was shown in (k). The HIFAL levels are shown in (l). m HIFAL activates the transcriptional activity of HIFAL promotor in a dose-dependent manner under hypoxia. HIFAL plasmid (0, 0.25, 0.5, 1 μg) was used for the transfection into MCF-7 cells. For b, f, j, p < 0.0001 versus IgG precipitation. Graphs show means ± SD of experimental triplicates, p values were determined by two-sided unpaired t-test (a–m). Source data are provided as a Source Data file.