Bioenergetic profiles of breast cancer cells with different expression of estrogen receptor (ER), progesterone (PR), and human epidermal growth factor-2 (HER2) using the XF24 analyzer. (A) Breast cancer cell-receptor status used in this study (left) and breast cancer cell lines with different expression of ER, PR, and HER2 and their respective oxygen consumption rates (OCR) and acidification rates (ECAR) (right). Group I (BT20, MDA468, MDA231, MDA436, SKBR3) was compared to ER/PR-positive cells or Group II (BT474, MCF7, T47D, ZR751) (50,000 cells/well for each cell line). Results are represented as average fold-change ± SD of at least three independent experiments. (B) Ratio of OCR/ECAR in breast cancer cells with different expression of ER, PR, and HER2. (C) Comparison of ATP levels in breast cancer cells between the triple-negative breast cancer (TNBC) group (BT20, MDA468, MDA231, MDA436) and ER/PR-positive group (BT474, MCF7, T47D, ZR751). (D) Comparison of NADH/NAD+ ratios in breast cancer cell lines between the TNBC (BT20, MDA468, MDA231, MDA436) and ER/PR-positive group (BT474, MCF7, T47D, ZR751). (E) Comparison of mitochondrial respiratory function in BT474 (triple-positive) and MDA468 (triple-negative) breast cancer cells. Oxygen consumption in BT474 (20, 000 cells/well) and MDA468 cells (50,000 cells/well) was analyzed using XF24 under basal conditions (cellular assay medium), following by sequential additions of oligomycin (10 ng/ml), CCCP (4 μM), and then rotenone (1 μM) as indicated. OCR and ECAR were normalized by cell number in each well at the end of the experiments. For optimal analysis, we used 50,000 for TNBC cells (MDA486 and MDA231) and 20,000 for ER-positive cells (BT474 and MVF7) in this assay. (F) MCE and SRC in TNBC cells (MDA231, MDA468) in comparison with that of breast cancer cells expressing ER and PR (MCF7, BT474). The bar graph represents mean ± SD from three experiments.