In vitro and ScRNA-Seq analysis of DNMT3A mutations in primary CD3⁺ T-cell cultures. (A, top) Representative Western blot of MYC-tagged DNMT3A. (A, bottom) Representative histogram of DNMT3A expression (MFI) and 5mC concentrations (MFI) in CD3⁺ T-cell cultures. (B) Representative flow cytometry plots for indicated cell types at listed time points. (C) Single-positive CD8⁺ T-cell percentages in vitro over indicated times in listed cell types (symbols represent mean ± standard error from n = 4 total donors covering n = 3 individual experiments) (D-E) UMAP (uniform manifold approximation and projection) plot of Seurat-identified clusters in CD3⁺ T-cell cultures. (E) Seurat-identified clusters annotated by SingleR for sample composition and immune cell identity. (F) Bar graph of the proportion of immune cell identities per sample as annotated by SingleR. (G) Violin plots of representative genes enriched in Seurat-identified clusters c0, c5, and c7. (H) Flow cytometry plots for listed cell types identifying CD8⁺ T_CM and T_EM cells. The gating strategy is shown graphically. Representative histograms, plots, and blots from (A-B) are taken from n = 3 independent experiments. Plots from H are from single-donor used for ScRNA-seq analysis. EV, empty vector; KD, knockdown; T_CM, T-central memory; T_EM, T-effector memory; T_reg, regulatory T cell.