Figure 2­(a) Extracellular acidification rate (ECAR) was measured in HE (n=7 biological replicates with 2-5 technical replicates each), EHT (n=7 biological replicates with 1-3 technical replicates each) and HSC-like (n=3 biological replicates with 2-4 technical replicates each) cells and glycolytic flux was assessed by extracellular flux analysis. Bar graphs show relative levels ± SEM of the indicated processes (from 7 (HE, EHT) or 3 (HSC-like) independent experiments, unpaired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (b) Dot plots showing gene expression levels of glycolytic enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). (c) FACS-sorted HE cells were subcultured with or without 2-DG (1 mM). Subculture day 3 representative FSC-A/CD43 plots are shown (n=7 biological replicates (d) Subculture day 3 representative GPA/CD43 plots and subculture day 6 CD45/CD43 plots are shown (n=6 and n=5 biological replicates (e) Subculture day 3 CellTrace Violet (CTV) fluorescence was assessed by flow cytometry (representative of n=4 biological replicates). (f) 2-NBDG uptake was measured by flow cytometry on day 10 for HE, EHT and HSC-like cells and mean MFI levels ± SEM are shown (n=4 biological replicates, paired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (g) Oxygen consumption rate (OCR) was measured in HE and EHT cells (n=3 biological replicates with 2-3 technical replicates each) and oxidative phosphorylation was assessed by extracellular flux analysis. Bar graphs show relative levels ± SEM of the indicated processes (from 3 independent experiments; unpaired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (h) TMRE fluorescence, with or without 100 µM FCCP treatment, was measured by flow cytometry on day 10 for HE, EHT and HSC-like cells and MFI - MFI FMO levels ± SEM relative to HE are shown (n=5 biological replicates, paired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (i) Basal OCR was measured in day 10 HE (n=6 biological replicates), EHT (n=5 biological replicates) and HSC-like (n=4 biological replicates) cells and bar graphs show mean levels ± SEM relative to HE (paired t-tests). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (j) Live cell imaging of HE and HSC-like cells stained with TMRE (red) at day 3 of subculture. Representative merged brightfield/TMRE and TMRE images are shown. Scale bars, 100 µm. Bar graphs show means of TMRE staining intensity from all replicate wells across all experiments (HE spindle and HE round, n=3 biological replicates with 3 technical replicates each; HSC-like, n=2 biological replicates with 3 technical replicates each, Kruskal-Wallis test with multiple comparisons). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (k) Dot plots showing gene expression levels of TCA cycle enzymes detected by scRNAseq and based on percent expressed (size of the dots) and average level of expression (color intensity). Data information: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001