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. 2019 Aug 29;47:329–340. doi: 10.1016/j.ebiom.2019.08.045

Fig. 7.

Fig. 7

cTnImAb1 treatment increases ENO1 expression followed by activation of PTEN and suppression of Akt signaling in mouse hearts.

a. Heat map from PathScan® Akt Signaling Antibody Array kit analysis showing changes in 16 phosphorylated proteins.

b & c. Statistical analysis of heat map data in a and volcano plot showing that cTnImAb1 treatment significantly increased PTEN Ser380 phosphorylation in the mouse heart. 1. PTEN Ser380, *p = .023, (t-test). 2. GSK-3b Ser9, *p = .038, (t-test). 3. PTEN Ser380, *p = .031 (t-test).

d&e. Immunoblot analysis showed that cTnImAb1 treatment significantly increased ENO1 expression and PTEN Ser380 phosphorylation but suppressed Akt Thr308 phosphorylation. β-actin served as a loading control. e is the statistical analysis of d. ENO1 *p = .002, pPTEN/PTEN/Actin, *p = .003, pAkt/Akt/Actin, *p = .003. (ANOVA analysis).

f-h. cTnImAb1 treatment (50 μg/ml) induced changes in ENO1 expression and phosphorylation of PTEN Ser380 and Akt Thr308 at different time points in cultured cardiomyoyctes. cTnImAb1 increased the p-Ser380-PTEN/total PTEN ratio at 8 h but decreased the pThr308-AKT/total Akt ratio at 8–12 h. Mouse IgG treatment did not alter pSer380 PTEN but significantly increased the pThr308-Akt/total Akt ratio at 24–36 h. f shows representative blot images of three independent experiments. g and h represent ImageJ-based densitometry analysis of relative changes in p-Ser380-PTEN/total PTEN ratio (G, 8 h *p = .020, 12 h *p = .024, t-test) and pThr308-AKT/total Akt (H, 12 h *p = .018, 24 h *p = .001, 36 h *p = .001, t-test).

i-l. Knockdown of ENO1 significantly suppressed cTnImAb1–induced alteration in PTEN/Akt signaling. Cultured cardiomyocytes were transfected with siRNA targeting Eno1 and then subjected to cTnImAb1 treatment (50 μg/ml) for 4–48 h as indicated. Mock-siRNA was used as a control. Depletion of Eno1 suppressed the p-PTEN Ser380/total PTEN ratio and increased the pAkt-Thr308/total Akt ratio at 4, 8, and 12 h, respectively. i shows representative blot images of three independent experiments. j represents ImageJ-based densitometry analysis of relative changes in ENO1 protein levels (J,48 h *p = .000, t-test). k and l represent ImageJ-based densitometry analysis of relative changes in p-Ser380-PTEN/total PTEN ratio (k, 48 h *p = .000, t-test) and pThr308-AKT/total Akt (l, 6 h *p = .000, 12 h *p = .000, t-test).