Fig 2: ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout (KO) suppresses calcineurin-induced cardiac hypertrophy. A, Heart weight/tibia length (HW/TL) ratio of ETS2F/F and KO mice crossed with calcineurin transgenic mice (CnA; n=8). B, Echocardiographic analyses of cardiac function of ETS2F/F and KO mice crossed with calcineurin transgenic mice. Left, M-mode echocardiographic images; right, fractional shortening (n=8). C and D, Histologic analyses of heart sections from ETS2F/F and KO mice crossed with calcineurin transgenic mice. Heart sections were stained with hematoxylin and eosin (C; scale bar, 2 mm) or wheat germ agglutinin (D; scale bar, 20 μm). E, Quantification of the relative cross-sectional area of the indicated groups (n=8). F, mRNA levels of hypertrophic marker genes (n=8). G, Western blot analyses and quantification of Rcan1.4 protein levels in indicated groups (n=6). *P<0.05; **P<0.01; ***P<0.001. ANP indicates atrial natriuretic peptide; βMHC, β-myosin heavy chain; BNP, brain natriuretic peptide; and Ctrl, control.

Fig 3: ETS2 is activated in hypertrophic hearts. A, Western blot analyses and quantification showing ETS2 phosphorylation and protein levels in mouse hearts at 1 or 3 weeks after sham operation or severe thoracic aortic constriction (sTAC; n=3). B and C, Western blot analyses (B) and quantification (C) of ETS2 phosphorylation and protein levels in cytoplasmic and nuclear fractions extracted from sham-operated control hearts and sTAC-induced hypertrophied hearts (n=3). D, ETS2 phosphorylation is increased in hearts from patients with dilated cardiomyopathy (DCM; n=8 for normal controls, n=19 for DCM). **P<0.01; ***P<0.001. ETS2 indicates a member of the E26 transformation-specific sequence (ETS) domain family; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant; and sTAC, severe transverse aortic constriction.

Fig 4: ETS2 regulates Foxa2 and NeuroD1 expression partly by inhibiting miR-124a expression. (A) Expression of FOXA2 and NEUROD1 was determined by immunoblotting in MIN6 cells transfected with negative control (NC) or miR-124a mimics, or incubated with EGF or not, or incubated with DMSO, LY294002, U0126 or gefitinib. (B) Expression of FOXA2 and NEUROD1 were determined by qRT-PCR in MIN6 cells following siETS2 or siNC transfection for 72 h. Subsequently, Western blotting was used to monitor the protein expression of FOXA2, NEUROD, phosphorylated ETS2 (Thr72) and total ETS2. ACTB (β-actin) was used as a loading control. (C) The same as in (B), but in MIN6 cells transfected with plasmids overexpressing wide-type (ETS2-WT) or mutant ETS2 (ETS2-T72A) or empty vector for control. (D) Expression of miR-124a, FOXA2 and NEUROD1 were determined by qRT-PCR and western blot in ETS2-overexpression or control MIN6 cells following miR-124a mimics or NC transfection. To restore miR-124a expression, MIN6 cells were co-transfected with the indicated combinations of mimics and vectors for 72 h. ACTB, the actin gene, internal control for cDNA detection. ACTB, actin, internal control for protein detection. Data represent the mean ± SE of three independent experiments (each n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 5: ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockdown attenuates phenylephrine-induced hypertrophy in vitro. A, Representative Western blots and quantification showing ETS2 phosphorylation and protein levels in isolated neonatal rat ventricular myocytes (NRVMs) treated with 50 μmol/L phenylephrine (PE) for 6 or 24 hours (n=6). B, Western blot analyses and quantification of ETS2 phosphorylation and expression in cytoplasmic and nuclear fractions extracted from PE-treated NRVMs (n=4). C, ETS2 phosphorylation and protein levels in NRVMs treated with PE or MEK inhibitor U0126, or both, for 24 hours (n=6). D, ETS2 was silenced in NRVMs by small interfering RNA (siRNA) transfection. E, Representative immunofluorescence images of α-actinin staining in NRVMs transfected with siRNA control (siNC) or siRNA targeting ETS2 (siETS2) and then treated with PE for 48 hours. Scale bar, 20 μm. F, Quantification of relative cardiomyocyte surface area (n=50). G, ETS2 knockdown inhibits PE-induced protein synthesis in NRVMs (n=6). H, mRNA levels of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), and βMHC (β-myosin heavy chain) in NRVMs transfected with siNC or siETS2 and then treated with PE for 24 hours (n=6). I, Rcan1.4 mRNA levels in NRVMs treated as in H (n=6). J, Chromatin immunoprecipitation (ChIP) analysis to detect ETS2 binding to the promoters of the indicated genes. ChIP was performed with an ETS2-specific antibody or immunoglobulin G control antibody in control NRVMs or NRVMs treated with PE or U0126, or both, for 6 hours. The occupancy of ETS2 at promoters is shown relative to background signals with immunoglobulin G control antibody (n=4). *P<0.05; **P<0.01; ***P<0.001. βMHC indicates β-myosin heavy chain; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; Ctrl, control; Cyto, cytoplasm; ETS2, a member of the E26 transformation-specific sequence (ETS) domain family; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant; and nucl, nucleus.