Fig 2: Disrupted COUP-TFII binding to FOXA1 by inhibitor.(A) MS analysis of CIA1 reduced COUP-TFII–binding proteins. Cells were treated with 4 µM CIA1 for 3 hours and then with biotin for 4 hours. IBAQ, intensity-based absolute quantification. (B) LNCaP cells with flag-COUP-TFII overexpression were treated with 4 µM CIA1 for 7 hours. IP was performed. (C) LNCaP cell growth was measured 4 days after with siRNA transfection. n = 3 per group. One-way ANOVA. (D) GSEA of FOXA1 signature with CIA1 regulated genes. FOXA1 signature was generated by GSEA37314. (E) LNCaP cells were transfected with siRNA for 72 hours. Western blot was performed, and qPCR was performed in (F). n = 3 per group. Two-way ANOVA. (G) LNCaP cells were treated with 4 µM CIA1 for 12 hours. ChIP-qPCR was performed. n = 3 per group. Two-way ANOVA. IgG, immunoglobulin G. (H) LNCaP cells were transfected with siRNA for 48 hours and then treated with 2 µM CIA1 for 18 hours. Western blot was performed, and qPCR was performed in (I). n = 3 per group. Two-way ANOVA. ns, P > 0.05; **P < 0.01 and ***P < 0.001.

Fig 3: COUP-TFII haploinsufficiency partially rescues calcineurin transgenic mice.(a) Experimental design. Echo, echocardiography. (b) Kaplan–Meier curve of mice with denoted genotypes over a 20-week observation period. (c) Left ventricular interior dimension at diastole at indicated time points. (d) Left ventricle relative wall thickness. Genotypes are denoted at bottom. N=10 (F+), 10 (Cre/F+), 5 (CnTg/F+) and 11 (CnTg/Cre/F+). *P<0.05; NS, P>0.05 (t-test) between denoted groups. Error bars denote the s.e.m.

Fig 4: Increased ROS production and compromised mitochondria functions.(a) Expression profile of affected mitochondrial genes in day 16 hearts. Blue marks downregulated and red depicts upregulated genes. (b–e) Day 9 hearts. (b) Activities of mitochondrial ETC enzymes normalized to citrate synthase (CS) activities. (c) Oxyblot analysis on mitochondria protein oxidation status. Hsp60 serves as loading control. (d) Activities of mitochondria aconitase at native and reduced forms, normalized to total mitochondria protein levels. N=3 for each genotype. (e) Mitochondria oxygen consumption rates. (f,g) Day 4 hearts. (f) Expression levels of key mitochondria ETC and ROS scavenger genes. N=6 (CTRL) and 10 (OE). (g) ChIP-qPCR results of COUP-TFII binding at genomic loci of ETC and ROS scavenger genes. N=3 for each genotype. RCR, respiratory control ratio. Oligo, effect of oligomycin. N=3 for each genotypes. *P<0.05; **P<0.01; ***P<0.001 (t-test) between CTRL and OE. Error bars denote the s.e.m.

Fig 5: Nr2f2 regulates ferroptosis and mitochondrial dysfunction via PGC-1a. (a) Representative Western blot of the PGC-1a protein level after Nr2f2 overexpression and knockdown (n = 4, means ± SDs, **p < 0.01 and ***p < 0.005, Student' t-test). (b) MDA level in each group (n = 6, means ± SDs, ***p < 0.005; ns: not significant, two-way ANOVA). (c) Representative Western blot of PTGS2 and GPX4. (n = 4, means ± SDs, *p < 0.05 and **p < 0.01, two-way ANOVA). (d) Lipid ROS was assessed by C11 BODIPY 581/591 staining coupled with flow cytometry analysis (n = 3, means ± SDs, ***p < 0.005, two-way ANOVA). (e) Cell viability was measured by CCK8 (n = 6, means ± SDs, ***p < 0.005, two-way ANOVA). (f) Representative JC-1 staining (scale bar = 50 µm, n = 6, means ± SDs; ns: not significant, ***p < 0.005, two-way ANOVA). (g) Intracellular ATP level in each group (n = 6, means ± SDs, ***p < 0.005, two-way ANOVA).