Fig. 7.

EGFL9 regulates cellular metabolism. a Visualization of EGFL9-COA3 interaction in vivo by BiFC live-cell imaging performed in 293 T cells. The top panels show 293 T cells transfected with p3xFlag-CMV-EGFL9-VN and p3xFlag-CMV-COA3-VC. The bottom panels show 293 T cells transfected with COA3-VC alone. Images of Venus (YFP), bright filed, and merge are shown. Scale bar: 20 µm. b BiFC result was confirmed by Co-IP western blot analysis. pLent-6-EGFL9-V5 and p3xFlag-CMV-COA3 constructs were co-transfected in 293 T cells. Co-IP was performed with anti-V5. Western blotting was then performed with anti-Flag antibody. c COX specific activity was measured using a Cytochrome Oxidase Activity Colorimetric Assay Kit. In all, 2.5 µg of mitochondria were mixed with reduced cytochrome oxidase and buffer per the manufacturer's instructions. The OD at 550 nm was measured and the maximum linear decay was used to calculate activity (units/mg). Each bar represents the mean ± SEM (standard error of the mean of a representative experiment performed in triplicate). P value was determined by unpaired two-tailed t-test. **P = 0.03. d Basic oxygen consumption rate (OCR) and maximum oxygen consumption rate were determined in a Seahorse bioanalyzer after normalization to cell numbers. FCCP, a potent uncoupler of mitochondrial oxidative phosphorylation, was added to both cell culture experiments. Several inhibitors of mitochondrial respiratory chain complex including Oligomycin, Rotenone, and Antimycin were also added. e Phosphorylation of COX was significantly increased in HMLE/EGFL9 cells in comparison with HMLE/LacZ cells. Mitochondrial protein was subject to IP with COX IV antibody and then blotted with COX II and pTyr antibodies. f Lactate levels in HMLE/LacZ and HMLE/EGFL9 culture medium were measured using a glycolysis cell-based assay. g The glucose concentration in HMLE/LacZ and HMLE/EGFL9 culture medium was measured with the Amplex Red Glucose Assay Kit. h, i Whole-cell reactive oxygen species (ROS) (h) and mitochondrial superoxide (i) were measured in HMLE/LacZ and HMLE/EGFL9 cells. For all experiments from f to i, each bar represents the mean ± SEM (standard error of the mean of a representative experiment performed in triplicate). P values were determined by unpaired two-tailed t-test. *P < 0.05; **P < 0.01, and ***P < 0.001