![Ascorbate deficiency promotes HSC and MPP quiescence. (A) Representative flow cytometry plots showing staining for CD229 and CD244 in HSCs and MPPs from Slc23a2-deficient or littermate control mice. These markers distinguish functionally distinct subsets of HSCs and MPPs that differ in terms of primitiveness.40 (B) Frequencies of HSC-1, HSC-2, MPP-1, MPP-2, and MPP-3 subsets based on these markers (a total of 6 mice per genotype from 4 independent experiments). (C-D) Analysis of GO biological process terms that were enriched among genes downregulated (fold change, <0.5; false discovery rate, <0.01) in Slc23a2-deficient vs control HSCs (C) or MPPs (D) by RNA sequencing. (E-G) Single-cell RNA sequencing (scRNA-seq) UMAP (Uniform Manifold Approximation and Projection) plots of CD48−LSK cells (which contain HSCs and MPPs) from Slc23a2-deficient and control mice, including the percentages of cells that were positive for the cell cycle genes Mki67 (F) and Cyclin d1 (G). (H-I) Incorporation of a 3-day (H) or 2-hour (I) pulse of BrdU into HSCs, MPPs, HPC1 cells, HPC2 cells, and all c-kit+ progenitors (a total of 4-18 mice per genotype from 3 [I] and 7 [H] independent experiments). (J) Representative H2B-GFP fluorescence in HSCs and MPPs from Slc23a2FL/FL;Col1a1-H2B-GFP;Rosa26-M2-rtTA mice without doxycycline treatment (gray) or after 6 weeks of doxycycline treatment (red HSCs, green MPPs). (K) Representative H2B-GFP fluorescence in HSCs and MPPs from Slc23a2-deficient or littermate control mice after 12 weeks of chase without doxycycline. (L) The percentages of H2B-GFPhigh, H2B-GFPlow, or H2B-GFPneg HSCs and MPPs after 12 weeks of chase without doxycycline (a total of 8 mice per genotype analyzed in 4 independent experiments). All data are presented as the mean ± standard deviation. Each dot represents a different mouse in panels B,H-I,L. Statistical significance was assessed using Student t tests with Holm-Sidak’s correction for multiple comparisons (B,H-I) or Mann-Whitney tests with Holm-Sidak’s corrections for multiple comparisons (L). All statistical tests were 2-sided (∗P < .05; ∗∗P < .01; ∗∗∗P < .001).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/145/1/10.1182_blood.2024024769/3/blood_bld-2024-024769-gr3.jpeg?Expires=1739624451&Signature=rVjinXXU--LwpkX-lBIsUJCxEBlLacwngRp0w-BnvdZNDgZldTL9LKm1UrmxGY5mfTAdBVfCdVuFfiR0nit0ZBxBiSmHg-0kAdFxzKczMotwLdUz1ZlD1WphfQeyg4UAj12-EFNgktduP0gezT1R8Qd6k7xv8UqBWyWAc6EBAWieSEHeKNcXGbomLsijD2DN~IJewVxXUGLbp4UgXpBJBghjPeG9MZ7IsHH2nJiSTgFitzbS5ti28wEIzIgEN0l5GvUD0EhlLWl~KOxELLTyyfZud9e~CfvUKuo79oRgWa6JNnYlmDFPs3FPgqNpf1-j4hW86e~9j62dMtdchLMaMQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Ascorbate deficiency promotes HSC and MPP quiescence. (A) Representative flow cytometry plots showing staining for CD229 and CD244 in HSCs and MPPs from Slc23a2-deficient or littermate control mice. These markers distinguish functionally distinct subsets of HSCs and MPPs that differ in terms of primitiveness.40 (B) Frequencies of HSC-1, HSC-2, MPP-1, MPP-2, and MPP-3 subsets based on these markers (a total of 6 mice per genotype from 4 independent experiments). (C-D) Analysis of GO biological process terms that were enriched among genes downregulated (fold change, <0.5; false discovery rate, <0.01) in Slc23a2-deficient vs control HSCs (C) or MPPs (D) by RNA sequencing. (E-G) Single-cell RNA sequencing (scRNA-seq) UMAP (Uniform Manifold Approximation and Projection) plots of CD48−LSK cells (which contain HSCs and MPPs) from Slc23a2-deficient and control mice, including the percentages of cells that were positive for the cell cycle genes Mki67 (F) and Cyclin d1 (G). (H-I) Incorporation of a 3-day (H) or 2-hour (I) pulse of BrdU into HSCs, MPPs, HPC1 cells, HPC2 cells, and all c-kit+ progenitors (a total of 4-18 mice per genotype from 3 [I] and 7 [H] independent experiments). (J) Representative H2B-GFP fluorescence in HSCs and MPPs from Slc23a2FL/FL;Col1a1-H2B-GFP;Rosa26-M2-rtTA mice without doxycycline treatment (gray) or after 6 weeks of doxycycline treatment (red HSCs, green MPPs). (K) Representative H2B-GFP fluorescence in HSCs and MPPs from Slc23a2-deficient or littermate control mice after 12 weeks of chase without doxycycline. (L) The percentages of H2B-GFPhigh, H2B-GFPlow, or H2B-GFPneg HSCs and MPPs after 12 weeks of chase without doxycycline (a total of 8 mice per genotype analyzed in 4 independent experiments). All data are presented as the mean ± standard deviation. Each dot represents a different mouse in panels B,H-I,L. Statistical significance was assessed using Student t tests with Holm-Sidak’s correction for multiple comparisons (B,H-I) or Mann-Whitney tests with Holm-Sidak’s corrections for multiple comparisons (L). All statistical tests were 2-sided (∗P < .05; ∗∗P < .01; ∗∗∗P < .001).
