Extended Data Fig. 8: BMAL1 does not interfere with HIF2A/HIF1B interaction or its transcriptional activity.
From: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury
a and b, RNA-seq analysis of HIF1B transcript levels in LV biopsies after aortic cross-clamping in morning vs. afternoon patients (a, n = 56 AM, n = 17 PM; boxplots show the 25th and 75th percentiles (box), median (central line), and minimum to maximum values (whiskers)) and in the AAR after 2 h of reperfusion at ZT8 or ZT20 in mice (b, n = 3 mice/time point; unpaired two-tailed t-tests). c-e, Hif1b transcript levels in the AAR on day 1 post-IRI in Bmal1loxP/loxP Myosin Cre+ and Myosin Cre+ mice (c, n = 5 Myosin Cre+ and n = 3 Bmal1loxP/loxP Myosin Cre+ mice; unpaired two-tailed t-tests), HIF1B protein levels by Western blot (d, n = 4 mice/group) and quantification (e, unpaired two-tailed t-tests). f-h, Hif1b transcript levels in mouse hearts 28 days post-Bmal1-AAV or control AAV injection (f, n = 3 mice/group; unpaired two-tailed t-tests), HIF1B protein levels by Western blot (g, n = 3 mice/group) and quantification (h, unpaired two-tailed t-tests). The same samples in (g) were also used in Supplementary Fig. 7c, sharing the β-actin blot. i-k, HIF1B transcript levels in HCMs transduced with Bmal1-AAV or control AAV under hypoxia (i, n = 3 independent experiments; unpaired two-tailed t-tests), HIF1B protein levels by Western blot (j, n = 3 independent experiments), and quantification (k; unpaired two-tailed t-tests). The same samples used in (j) were also used in Extended Data Fig. 7m, sharing the β-actin blot. l-n, HIF1B transcript levels in HCMs transduced with shBmal1-AAV or shControl AAV (l, n = 3 independent experiments; unpaired two-tailed t-tests), HIF1B protein levels by Western blot (m, n = 3 independent experiments), and protein quantification (n; unpaired two-tailed t-tests). The same samples in (m) were also used in Extended Data Fig. 7p, sharing the β-actin blot. o and p, Immunoprecipitation using HIF1B and HIF2A antibodies in HEK293 cells transfected with BMAL1-Flag or control plasmid under hypoxia or normoxia, analysed by Western blot (o, n = 3 independent experiments) and normalized to α-tubulin (p, one-way ANOVA). q and r, ChIP-qPCR using HIF1B antibody showing binding to the human EPO (q) and AREG (r) promoters under normoxia and hypoxia. n = 8 independent experiments; two-way ANOVA. s, Luciferase assays in HEK293 cells transfected with BMAL1-Flag (0–200 nM), HIF2A-HA, and a luciferase reporter for the human PGK1 promoter. n = 4 independent experiments; one-way ANOVA. All data are mean ± s.e.m.
