Extended Data Fig. 7: BMAL1 stabilizes HIF2A protein.
From: BMAL1–HIF2A heterodimer modulates circadian variations of myocardial injury
a and b, HIF2A transcript levels from RNA-seq of LV biopsy samples after aortic cross-clamping in morning and afternoon cardiac surgery patients (a, n = 56 morning, n = 17 afternoon; boxplots show the 25th and 75th percentiles (box), median (central line), and minimum to maximum values (whiskers)) and in the AAR of mice after 2 h of reperfusion at ZT8 or ZT20 (b, n = 3 mice/time point; unpaired two-tailed t-tests). c, Hif2a transcript levels by real-time PCR after 2 h of reperfusion in the AAR subjected to IRI at ZT8 or ZT20. n = 3 mice/time point; unpaired two-tailed t-tests. d-f, Hif2a transcript levels by real-time PCR in the AAR after 2 h of reperfusion in Bmal1loxP/loxP Myosin Cre+ mice and Myosin Cre+ mice (d, n = 5/Myosin Cre+ and n = 3/Bmal1loxP/loxP Myosin Cre + ; unpaired two-tailed t-tests). HIF2A protein levels by Western blot in the nuclear fractions (e, n = 3/Myosin Cre+ and n = 5/Bmal1loxP/loxP Myosin Cre + ) and quantification (f, Welch’s t-tests). g-k, C57BL/6J mice treated with NOB (200 mg/kg, i.p.) and HIF2A protein levels were assessed by Western blot (g, h, n = 3 mice/group/time point; two-way ANOVA) or transcript levels by real-time PCR (i, n = 4 mice/Veh-treated/ZT8, n = 4 mice/Veh-treated/ZT20, n = 4 mice/NOB-treated/ZT8, and n = 3 mice/NOB-treated/ZT20; two-way ANOVA). Immunostaining of HIF2A in the myocardium (j, n = 4 mice/group/time point, scale bar, 25 μm) and quantification (k, two-way ANOVA). l-q, HCMs transduced with Bmal1-AAV or shBmal1-AAV were treated with 1% O2 for 4 h. BMAL1 and HIF2A transcript levels by real-time PCR (l, o), protein levels by Western blot (m, p), and quantification (n, q). n = 3 independent experiments; Unpaired two-tailed t-tests were used for all comparisons, except for BMAL1 protein levels in (n), which were analysed using Welch’s t-tests. The samples used in (m) were also used in Extended Data Fig. 8j, sharing the β-actin blot. The samples used in (p) were also used in Extended Data Fig. 8m, sharing the β-actin blot. r, ChIP-qPCR of BMAL1 binding to E-box elements in the human HIF2A promoter under normoxic and hypoxic conditions. n = 4 independent experiments; one-way ANOVA. s-v, HCMs transduced with Bmal1-AAV (s, t) or shBmal1-AAV (u, v) were exposed to 1% O2 for 4 h and treated with CHX to assess HIF2A degradation. Protein levels were quantified and plotted to determine half-life using exponential decay. n = 3 independent experiments. w, x, HEK293 cells transfected with HIF2A-Myc, Ub-HA, and BMAL1-Flag, treated with MG132, and subjected to HIF2A immunoprecipitation. Ubiquitination levels were analysed (w) and quantified (x). n = 3 independent experiments; one-way ANOVA. All data are mean ± s.e.m.
