ATF4 loss alters transcriptional lineage commitment in HSPCs. (A) Experimental workflow showing the strategy used to sort cells from the BM of fl/fl and Δ/Δ mice (n = 3 mice) for scRNA-seq. (B) Uniform manifold approximation and projection (UMAP) visualization of 14 189 BM lin–cKit+ (LK+) cells categorized into 15 clusters from the fl/fl and Δ/Δ mice. (C) UMAP plot showing the cell subset distribution frequencies within the fl/fl and Δ/Δ BM LK+ cell populations. (D) Transcriptome cluster frequency changes in Atf4–depleted LK+ cells relative to the control. ∗∗P < .01; ∗∗∗P < .001; χ2 test. (E) UMAP plots colored according to genotype (fl/fl: blue, 1544 cells; Δ/Δ: red, 117 cells; left). The fourth principal component, which had the strongest correlation with erythrocyte maturation, was used to construct an erythrocyte maturation score. Each dot represents a single cell. Distribution of cells according to erythrocyte maturation scores (right). ∗∗∗P < .001; Wilcoxon rank-sum test. (F) Barplot showing the fate bias of single HSC/MPPs computed by FateID. ns, not significant; ∗∗P < .01; ∗∗∗P < .001; 2-sided Fisher exact test. (G) UMAP visualization of 19 706 CMP cells from fl/fl and Δ/Δ mice categorized into 8 clusters. (H) UMAP plot showing the cell subset distribution frequencies in the fl/fl and Δ/Δ CMP cell populations. (I) Transcriptome cluster frequency changes in Atf4-depleted CMP cells relative to the control. ∗P < .05; ∗∗∗P < .001; χ2 test. (J) Cell cycle distributions of Ery1 and Ery2 in fl/fl and Δ/Δ LK+ cells. (K) Violin plots showing the expression of the G1/S phase transition gene set in Ery1 and Ery2 cells from fl/fl and Δ/Δ group. (L) Violin plots showing the expression level of the DNA replication gene set in Ery1 and Ery2 cells from fl/fl and Δ/Δ groups. ns, not significant; ∗∗∗P < .001; Wilcoxon rank-sum test. (M) Gene set enrichment analysis (GSEA) of DNA replication in Ery1 cells from the fl/fl and Δ/Δ groups. (N) Expression levels of representative genes related to DNA replication in Ery1 subsets within fl/fl and Δ/Δ LK+ cell populations. (O). Violin plots showing the expression of the replication stress gene set in Ery1 and Ery2 cells from fl/fl and Δ/Δ groups. (P) GSEA of replication stress in Ery1 cells from the fl/fl and Δ/Δ groups. (Q) GSEA of DNA damage in Ery1 cells from fl/fl and Δ/Δ groups. (R) Violin plots showing the expression of the DNA repair gene set in Ery1 and Ery2 cells from the fl/fl and Δ/Δ groups. (S) Violin plots showing the expression of the apoptosis gene set in Ery1 and Ery2 from the fl/fl and Δ/Δ groups. (T) GSEA of apoptosis in Ery1 cells from the fl/fl and Δ/Δ groups. ns, not significant; ∗P < .05; ∗∗∗P < .001; Wilcoxon rank-sum test.

ATF4 loss alters transcriptional lineage commitment in HSPCs. (A) Experimental workflow showing the strategy used to sort cells from the BM of fl/fl and Δ/Δ mice (n = 3 mice) for scRNA-seq. (B) Uniform manifold approximation and projection (UMAP) visualization of 14 189 BM lin–cKit+ (LK+) cells categorized into 15 clusters from the fl/fl and Δ/Δ mice. (C) UMAP plot showing the cell subset distribution frequencies within the fl/fl and Δ/Δ BM LK+ cell populations. (D) Transcriptome cluster frequency changes in Atf4–depleted LK+ cells relative to the control. ∗∗P < .01; ∗∗∗P < .001; χ2 test. (E) UMAP plots colored according to genotype (fl/fl: blue, 1544 cells; Δ/Δ: red, 117 cells; left). The fourth principal component, which had the strongest correlation with erythrocyte maturation, was used to construct an erythrocyte maturation score. Each dot represents a single cell. Distribution of cells according to erythrocyte maturation scores (right). ∗∗∗P < .001; Wilcoxon rank-sum test. (F) Barplot showing the fate bias of single HSC/MPPs computed by FateID. ns, not significant; ∗∗P < .01; ∗∗∗P < .001; 2-sided Fisher exact test. (G) UMAP visualization of 19 706 CMP cells from fl/fl and Δ/Δ mice categorized into 8 clusters. (H) UMAP plot showing the cell subset distribution frequencies in the fl/fl and Δ/Δ CMP cell populations. (I) Transcriptome cluster frequency changes in Atf4-depleted CMP cells relative to the control. ∗P < .05; ∗∗∗P < .001; χ2 test. (J) Cell cycle distributions of Ery1 and Ery2 in fl/fl and Δ/Δ LK+ cells. (K) Violin plots showing the expression of the G1/S phase transition gene set in Ery1 and Ery2 cells from fl/fl and Δ/Δ group. (L) Violin plots showing the expression level of the DNA replication gene set in Ery1 and Ery2 cells from fl/fl and Δ/Δ groups. ns, not significant; ∗∗∗P < .001; Wilcoxon rank-sum test. (M) Gene set enrichment analysis (GSEA) of DNA replication in Ery1 cells from the fl/fl and Δ/Δ groups. (N) Expression levels of representative genes related to DNA replication in Ery1 subsets within fl/fl and Δ/Δ LK+ cell populations. (O). Violin plots showing the expression of the replication stress gene set in Ery1 and Ery2 cells from fl/fl and Δ/Δ groups. (P) GSEA of replication stress in Ery1 cells from the fl/fl and Δ/Δ groups. (Q) GSEA of DNA damage in Ery1 cells from fl/fl and Δ/Δ groups. (R) Violin plots showing the expression of the DNA repair gene set in Ery1 and Ery2 cells from the fl/fl and Δ/Δ groups. (S) Violin plots showing the expression of the apoptosis gene set in Ery1 and Ery2 from the fl/fl and Δ/Δ groups. (T) GSEA of apoptosis in Ery1 cells from the fl/fl and Δ/Δ groups. ns, not significant; ∗P < .05; ∗∗∗P < .001; Wilcoxon rank-sum test.

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