Fig 2: Mitochondrial gene expression and function after four exposures. Mitochondrial electron transport chain maximal activities defined as the amount of substrate consumed per minute per ng or mg of protein for A complex I, B complex II, C complex III, D complex IV, and E complex V at day four exposure (n = 4, each group). F Using Ingenuity Pathway Analysis (IPA), mitochondrial genes that were differentially expressed in the oxidative phosphorylation pathway are displayed for co-exposure. Genes colored red have statistically increased transcription, genes colored green have statistically decreased transcription, and genes colored grey have no change in transcription, compared to the air control group. The shape of the genes is not indicative of function or pathology. Coloring of the entire complex, i.e. complex I, II, III, IV, and V, indicates global increase/decrease of expression of the entire complex. F The maximal activities of ETC complexes I–V are displayed above each of the corresponding complex units. G Western blot of ATP5F1 and quantification. H Electron flow was measured in co-exposure compared to the sham group for complexes I–IV. I Total ATP content was quantified by day and group for CB, O3, and CB + O3 groups. Day 1 (n = 4, each group) and Day 4 (n = 4, each group). Differences between groups were considered statistically significant if P ≤ 0.05, denoted by *. Data are presented as the mean ± standard error of the mean (SEM), when appropriate. Sham—4 = filtered air exposed for 4 days, CB—4 = 10 mg/m3 CB exposure for 3 h repeated four times (24 h apart), O3—4 = 2 ppm O3 exposure for 3 h repeated four times (24 h apart), CB + O3—4 = 10 mg/m3 CB and 2 ppm O3 inhalation co-exposure for 3 h repeated four times (24 h apart), OCR oxygen consumption rate