Fig. 3

T3 modulates hESC metabolism and gene expression. A, B T3 modulates hESC mitochondrial respiration. H1 cells treated with E8 or E8 + T3 (500 nM) for 3 days were seeded into the assay plate, cultured for one more day with the same treatment and subjected to the test with T3 added into the assay medium in corresponding wells. Oxygen consumption rate (OCR) of H1 hESCs measured with Mito Stress Test using Seahorse XFe96 analyzer (A), basal respiration, maximal respiration and ATP Synthase-Associated Respiration calculated from the assay results in panel A (B). n ≥ 3, *P < 0.05. C Heatmap of intracellular levels of metabolites quantified by LC–MS/MS. Data are shown as log2 fold change of the peak area of corresponding metabolite normalized to the control group (E8). n = 5. Red color indicates upregulation and blue downregulation. Gray represents the compound was not detected in the assay. D Heatmap of amino acids consumption in culture medium, quantified by LC–MS/MS. Data are shown as Z-Score of the peak area of corresponding metabolite. n = 5. Red color indicates more secretion/less consumption, while blue color indicates less secretion/more consumption. E RNA-seq analysis of the effect of T3 in high-density (HD, > 90% confluence) and low-density (LD, < 70% confluence) H1 culture. Cells were treated with or without T3 (500 nM) for 3 days. Left, heatmap of RNA-seq DEG. Right, GO term analysis based on clustered genes. F T3 reduces ROS level in hESCs. hESC ROS level was detected by CellROX green kit. Scale bar, 100 μm