Fig. 4: Tox4 loss impairs proliferation of the DP blast population within DP cells.

a Combined UMAP plot showing clusters of all cells from thymi of 2 pairs of control and cKO mice. b Two-dimensional representation of αβ T cells via UMAP, as colored by the Monocle3 estimated pseudotime. Each dot represents one cell, and black lines represent Monocle3 estimated developmental trajectory. c Boxplot of Monocle3 predicted pseudotime of αβ T cells colored by cluster. Black dots represent median pseudotime values of cell clusters, and they are connected with black lines to manifest the increasing median pseudotime of cell clusters along the estimated developmental trajectory. A small cluster, 10_DPre, was skipped for straying far away from the trendline. The width of each box is proportional to the size of that cluster. d Cell count of each cluster relative to that of DN cells (cluster 01_DN). The relative cell count of DN cells for each biological replicate is set as 1. e Boxplots with beeswarm plots of proliferation scores calculated by averaging expression of a set of proliferation signature genes for each cluster in control and Tox4 cKO mice. f–h Heatmaps showing log2 fold change of differentially expressed genes of 02_DPbla1 (f), 03_DPbla2 (g), and 04_DPre1 (h) clusters in Tox4 cKO thymocytes relative to control thymocytes. i Violin plots showing expression of cell-cycle-related differentially expressed genes of 02_DPbla1, 03_DPbla2, and 04_DPre1 clusters. j–l Violin plots comparing expression level of downregulated and unaffected genes upon Tox4 loss within 02_DPbla1, 03_DPbla2, and 04_DPre1 clusters. For boxplots in (c, e, and j–l), the standard boxplot notation was used (lower/upper hinges–first/third quartiles; whiskers extend from the hinges to the largest/smallest values no further than 1.5 x inter-quartile ranges; middle line–the median). Data beyond the end of the whiskers are called “outlying” points and are plotted individually in (c). Two independent experiments were performed (n = 2). The differences of proliferation score between Tox4 cKO and control cells (e) and the differences of expression between two groups of genes (j–l) were tested using two-sided Wilcoxon rank-sum test. NS: P ≥ 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.