Fig. 5: Metabolic heterogeneity in initial cells.

a Distribution and box plot (inset) of DCFDA intensity in initial cells and cells under OS. Data in distribution plot was z scored. Variance was indicated by IQR and MAD values in Supplementary Table 1. W = 58627, P < 2.2e-16 in unpaired two-tailed Wilcox rank sum test. The data presented in the inset was normalized to the values of initial cells. Box plots extend from 25th to 75th percentiles; central lines represent medians; whiskers extend over 1.5 times the interquartile range (IQR, the distance from 25th to 75th percentile); dots represent outliers. For single-cell DCFDA intensity, a total of 1740 initial cells (blue) and 960 oxidative stressed cells (gray) from 3 independent experiments were analyzed. b UMAP visualization of metabolic subtypes in initial cells. Green: cells of Cluster-I (n = 43). Purple: cells of Cluster-II (n = 183). c Heatmap of potential metabolic markers in Cluster-I and Cluster-II. The data was z score scaled. The color represents relative abundance of metabolites. Yellow: relatively high abundance; green: relatively low abundance. Each row represents a metabolite and each column represents a cell. The representative metabolites are labeled on the left and the clusters are labeled on the top. ATP: adenosine triphosphate; GSH: glutathione; GSSG: oxidized glutathione; Hypt: hypotaurine; O-PE: O-phosphoethanolamine; UTP: uridine triphosphate. d A heatmap illustrating the metabolic similarity between subtypes of initial cells (Cluster-I/II) and subtypes of oxidative stressed cells (C1 to C6). Each row represents an initial cell (subtypes are labeled on the left) and each column represents an oxidative stressed cell (subtypes are labeled on the top). The heatmap was plotted with similarity (1/distance) and the data was z score scaled. The distance between cells was calculated based on single-cell metabolome. The color represented the relative similarity: red, relative high similarity; blue, relative low similarity. Cells of Cluster-I is more similar to the cells with lower OS levels (cells of C1 and part of C2). e Enrichment of GSH in cells of Cluster-I visualized on the UMAP plot. Each dot represents a cell, the color of the dots represents the relative GSH abundance. Red: relatively high abundance; blue: relatively low abundance. Data was z score scaled. f Quantification of GSH abundance in Cluster-I (n = 43, green) and Cluster-II (n = 183, purple). Data was normalized to values in Cluster-I. Data is represented as mean ± s.e.m. Data were collected from at least three biological replicates. W = 7843, P = 5.85e−43 in unpaired two-tailed Wilcox rank sum test. g The correlation of every two metabolites were calculated (reflected as Pearson’s r) and the network was constructed based on the correlation data for initial cells belonging to Cluster-I (left) and Cluster-II (right) subtypes. In the network, each node represents a metabolite while an edge connecting two nodes indicates their correlation. The big green dot denotes GSH and the small blue dots denote GSH correlated metabolites. Blank-colored dots indicate metabolites without any correlation to GSH. h Rewiring score calculated with DyNet algorithm. Metabolites with higher scores were more rewired in topology in the correlation network of initial cells. For (a, b, e, f), Source data are provided as Source Data files. GSH: glutathione.