Figure 7.

Knockdown of ETFα in HUVECs affects mitochondrial bioenergetics and oxygen consumption. (a) Bioenergetic profile of control HUVECs measured by the Seahorse XF analyzer, in which the oxygen consumption rate (OCR) over time is determined. BSA was used to assess utilization of endogenous fatty acid; Palm-BSA was used to assess utilization of exogenous fatty acid. Etomoxir is an inhibitor of fatty acid oxidation (FAO). Oligomycin, an ATP synthase (complex V) inhibitor, was added to detect cellular ATP production; FCCP, a potent uncoupler of oxidative phosphorylation in mitochondria, was added to assess maximal respiration of HUVECS; a mixture of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor, was included to measure nonmitochondrial respiration in cells. (b) OCR profiles in ETFα knockdown HUVCEs; (c) basal respiration of control and ETFα knockdown HUVECs. OCRs in different groups were analyzed with one-way ANNOVA, ^∗∗∗∗P < 0.00001, n = 10; (d) maximal respiration of control and ETFA knockdown HUVECs. OCRs in different groups were analyzed with one-way ANNOVA, ^∗∗∗P < 0.0001, ^∗∗∗∗P < 0.00001, n = 10; (e) representative immunoblot for HIF1α and ETFα in control-, scramble shRNA-, and ETFA shRNA lentivirus treated HUVECs. Cell lysates were collected at 24 hours or 48 hours after lentivirus transfection. Acetate was added to ETFα knockdown HUVECs. β-Actin was the loading control; (f) representative immunoblot for HIF1α and ETFα in control-, scramble shRNA-, and ETFA shRNA lentivirus-treated HUVECs. HUVECs were exposed to hypoxic condition for 12 hours before collection. Cell lysates were collected at 24 hours or 48 hours after lentivirus transfection.