Fig. 5: CircDhx32 suppresses the transcription of AdipoR1 by competitively binding to FOXO1.

a RIP assay showing the increased interaction capacity between circDhx32 and FOXO1 under H/R conditions. n = 3. ^**P < 0.01 vs. Ctrl. P values were determined via an unpaired t test. b, c Western blot analysis of the nuclear and cytoplasmic protein expression of FOXO1 upon circDhx32 silencing. n = 5‒6. ^**P < 0.01 vs. NC. P values were determined via an unpaired t test. d FISH-IF assay showing the colocalization of circDhx32 (red) and FOXO1 (green) after H/R. DAPI (blue) was used to stain the nuclei. Scale bar = 20 μm. n = 4. e, f Western blot analysis of AdipoR1 in I/R-treated mouse hearts and H/R-induced cardiomyocytes. n = 6. ^**P < 0.01 vs. Sham, ^**P < 0.01 vs. Ctrl. P values were determined via an unpaired t test. g Relative mRNA level of AdipoR1 after transfection with si-NC or si-FOXO1. n = 5‒6. ^**P < 0.01 vs. NC. P values were determined via an unpaired t test. h The protein expression of AdipoR1 after FOXO1 silencing. n = 6. ^**P < 0.01 vs. NC. P values were determined via an unpaired t test. i ChIP‒qPCR was performed to analyse the binding of FOXO1 to the AdipoR1 promoter upon circDhx32 overexpression. IgG was used as a negative control. n = 6. ^**P < 0.01 vs. NC. P values were determined via an unpaired t test^. j Relative luciferase activity was determined in NMVCs cotransfected with a luciferase reporter plasmid containing the AdipoR1 promoter sequence and an overexpression plasmid containing circDhx32 and FOXO1. n = 3. ^**P < 0.01 vs. NC; ^#P < 0.05, ^##P < 0.01 vs. oe-FOXO1. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed to evaluate significant differences. k RNA EMSAs were used to detect the interaction between AdipoR1 and FOXO1 in primary cardiomyocytes with or without circDhx32 plasmid transfection. l Relative AdipoR1 mRNA expression was examined by qRT‒PCR in cardiomyocytes cotransfected with si-FOXO1 and si-circDhx32. n = 5‒6. ^**P < 0.01 vs. si-NC; ^##P < 0.01 vs. si-FOXO1. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed to evaluate significant differences. m The protein level of AdipoR1 was determined by Western blotting. n = 5. ^**P < 0.01 vs. si-NC; ^##P < 0.01 vs. si-FOXO1. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed to evaluate significant differences. n, o mRNA and protein levels of AdipoR1 in cardiomyocytes after overexpression of FOXO1 and circDhx32. n = 6. ^**P < 0.01 vs. oe-NC; ^#P < 0.05, ^##P < 0.01 vs. oe-FOXO1. One-way ANOVA followed by Dunnett’s multiple comparisons test was performed to evaluate significant differences.