Figure 7. Depletion of Bmal1 alters metabolic gene pathways, causing an increase in respiration and mitochondrial ROS production in embryonic stem cells (ESCs).
(A) Heat map of the expression of tricarboxylic acid cycle (TCA) and mitochondrial complex I–V genes from wild-type (Bmal1 WT) and Bmal1 KO ESCs RNA-seq. (B) Basal glycolysis levels determined as extracellular acidification rate (ECAR) in wild-type (WT) and Bmal1 KO ESCs (n = 3). (C) Bioenergetics assays in ESCs from WT and Bmal1 KO ESCs. The different drugs used in the assay are indicated in grey (n = 3). (D) OCR/ECAR under basal conditions for the indicated cell lines at three different time points (n = 3). (E) Proton leak shown as OCR in WT and Bmal1 KO ESCs (n = 3). (F) Fluorescence representative microscopy images showing MitoSOX fluorescence (red) in WT and Bmal1 KO ESCs. Nuclei were stained with DAPI and shown in blue. The scale bar represents 50 μm. (G) Box plots showing the mean fluorescence intensity of MitoSOX treated Bmal1 WT and KO cells. Center lines, medians; whiskers extend to the 10th/90th percentile. (H) Relative expression of the indicated genes in Bmal1 WT and KO cells with or without antimycin A/rotenone (AA/Rot) treatment. t test was performed. (n = 3). In (B, C, D, E, H), data are represented as mean ± SD. Two-tailed unpaired t test was performed. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; AA, Antimycin A; FCCP, carbonyl cyanide-4 phenylhydrazone; ns, not significant; OCR, oxygen consumption rate.
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