Fig. 3: GPAT4 regulates ER homeostasis and ER-Mito communications in the endocardium.
A RNA-seq analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, Heatmap of dysregulated genes. Red and blue colors represent upregulated and downregulated genes, n = 3. B qPCR analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, ***P = 0.0005, **P = 0.0010, ***P = 0.0002, ***P = 0.0007, ***P = 0.0002, ***P = 0.0006, ***P = 0.0007. C, D Western blotting analysis and quantification of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, **P = 0.0014, ***P = 0.0004, **P = 0.0015, **P = 0.0013, *P = 0.0106. E, F Immunoprecipitation-mass spectrometry (GPAT4 antibody) analysis with KEGG/GO (HUVECs). G The association between GPAT4 protein and EIF2S1/eIF2α protein predicted by AF3. H Immuno-precipitation (GPAT4 and EIF2S1) and Western blotting analysis (HUVECs). I AF3 modeling of the association relationship among the three proteins of PERK, GPAT4 and EIF2S1/eIF2α. J AF3 modeling of association between PERK protein and EIF2S1/eIF2α protein. K Immuno-precipitation (PERK and EIF2S1) and Western blotting analysis in Ctrl and siGPAT4 groups (HUVECs). L Immuno-precipitation (PERK and EIF2S1) and Western blotting analysis in Ctrl and GPAT4 OE groups (HUVECs). M, N Ctrl and GPAT4 OE groups, Western blotting analysis and quantification (HUVECs), n = 3. Left-Right, **P = 0.0081, **P = 0.0012, *P = 0.0181, **P = 0.0016, **P = 0.0013. O, P TEM analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F endocardial cells in E11.5 hearts and quantification of ER-Mito contacts, n = 3. Scale bars, 1 µm. Left-Right, *P = 0.0161, *P = 0.0158, *P = 0.0495, **P = 0.0078. Q, R Western blotting analysis and quantification of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, *P = 0.0168, **P = 0.0042, ***P = 0.0004, **P = 0.0013, **P = 0.0036. Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test.
