5-FU stress accelerates Atf4-deletion–induced BM hematopoietic failure. (A) Experimental workflow for sorting of CMP cells from the BM of fl/fl and Δ/Δ mice for ribosome profiling (Ribo-seq). (B) Plots showing the DEGs identified by Ribo-seq analysis in CMP cells from fl/fl vs Δ/Δ mice. (C) GSEA plot of ribosome biogenesis based on Ribo-seq data from CMP cells in the fl/fl and Δ/Δ groups. (D) GSEA plot of ribosome biogenesis based on RNA-seq data from CMP cells of the fl/fl and Δ/Δ groups. (E) GSEA plot of PreCFU-E based on Ribo-seq data from CMP cells of the fl/fl and Δ/Δ groups (left). Relative changes in translation efficiency for selected transcripts from the PreCFU-E gene set are shown in blue, and relative changes in messenger RNA expression are shown in red (right). (F) Sorted cell populations from fl/fl and Δ/Δ mice were treated with puromycin and then analyzed by western blotting using antibodies against the indicated proteins. (G) Representative polysome profiles from cKit⁺ cells of fl/fl and Δ/Δ mice. The absorption profile of a linear sucrose gradient at 254 nm is depicted, with sedimentation and major ribosomal peaks indicated. (H) Survival curves of fl/fl and Δ/Δ mice treated with a single dose of 5-FU. (I) BM cell numbers in fl/fl and Δ/Δ mice 10 days after 5-FU treatment (n = 3-4 mice). (J) Number of LT-HSCs in the BM of fl/fl and Δ/Δ mice 10 days after 5-FU administration (n = 3 mice). (K) Routine blood parameters of fl/fl and Δ/Δ mice at the indicated time points after 5-FU treatment (n = 4-9 mice). (L) cKit⁺ cells from the BM of fl/fl and Δ/Δ mice were given 5-FU and then 10 days later treated with puromycin and analyzed by western blotting using antibodies against the indicated proteins. Data represent the mean ± SD. ∗∗P < .01; ∗∗∗P < .001; unpaired 2-tailed Student t test.