Analysis of mitochondrial respiratory complex activity and expression of molecules involved in energy metabolism in triple-negative breast cancer (TNBC) cells in comparison with other subtypes of breast cancer cells. (A) Analysis of mitochondrial respiratory complex activity in breast cancer cells with different receptor status with the Clark electrode using the oxythem system as described in Methods. The representative oxygen consumption curves for BT474 cells (triple-positive) and MDA231 (triple-negative) are shown on the top panel; the quantitative data for each segment of the mitochondrial respiratory chain activity in four different cell lines (two triple-negative lines: MAD231 and MDA468; two receptor-positive lines: BT474 and MCF7) are shown in the lower panel. Data are mean ± SD (n = 3). (B) Expression of mitochondrial respiratory chain components in breast cancer cells. The protein levels of complex I subunit NDUFB8 (ND6), complex II 30 kDa subunit, complex III Core 2 subunit, Complex IV subunit II, and the ATP synthase subunit α, cytochrome c (cyt c) and SCO2 were determined by western blot analysis. A representative blot from at least three independent experiments is shown. (C) Expression of PDH, HKII, HKI, Glut-1, Glut-4, LDH-A, PKM2, total Akt and phospho-Akt (ser 473) in breast cancer cells determined by western blot. Data are representative of three independent experiments. (D) Analysis of PDK1 mRNA expression in breast cancer cells by RT-PCR. Data are representative of three independent experiments. (E) Analysis of p70S6K and AMPK status in breast cancer cells by western blot. One representative blot from three independent experiments is shown. (F) Analysis of p70S6K expression in breast cancer cells by RT-PCR. Data are representative of three independent experiments.