Figure 1.
Reduced frequency and loss of quiescence of HSCs in th3 mice. (A) Representative plots of HSCs gated from BM Lin−Sca-1+c-Kit+ cells. WT and th3 HSC frequency on BM Lin− cells and absolute counts from both femurs and tibiae. WT, n = 8; th3, n = 9. (B) Evaluation of cell cycle distribution among G0/G1, S, and G2/M phases of WT and th3 HSCs by cytofluorimetry after 7-AAD staining. 7-AAD/Ki-67 staining was performed to discriminate between G0 and G1 phases. WT, n = 9; th3, n = 8. (C) Frequency of cycling HSCs expressed as a percentage of LRCs, revealing BrdU+ HSCs by in vivo BrdU pulse-chase assay. Chase fold-decrease, compared with respective pulse labeling is reported. WT pulse, n = 3; WT chase, n = 5; th3 pulse, n = 3; th3 chase, n = 3. (D-E) GSEA enrichment plots of the cell cycle–associated categories from the biological process branch of the GO database, showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list. Enrichment of th3 HSCs vs WT HSCs is reported. (F) Representative images of γH2AX+ foci in the nuclei of WT and th3 Lin− cells. Analysis of the frequency of cells accumulating DNA damage, shown as a percentage of γH2AX+ cells, is reported. WT, n = 3; th3, n = 3. Immunofluorescence analysis by anti-phospho-H2AX (Ser139) staining was performed. Cell nuclei were stained with Hoechst 33342. (G) GSEA enrichment plot of the cellular response to stress category from the Reactome Pathway database, showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list. Enrichment in th3 HSCs vs WT HSCs is reported. (H) GSEA enrichment plot showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list of key genes reported to be associated with HSC stemness. Enrichment in th3 HSCs vs WT HSCs is reported. (I) Methylcellulose CFU replating assay using sorted HSCs from WT and th3 mice. Absolute number of CFUs per plate is shown at different platings. WT, n = 6; th3, n = 6. *P < .05; **P < .01. NES, normalized enriched score.

Reduced frequency and loss of quiescence of HSCs in th3 mice. (A) Representative plots of HSCs gated from BM Lin−Sca-1+c-Kit+ cells. WT and th3 HSC frequency on BM Lin− cells and absolute counts from both femurs and tibiae. WT, n = 8; th3, n = 9. (B) Evaluation of cell cycle distribution among G0/G1, S, and G2/M phases of WT and th3 HSCs by cytofluorimetry after 7-AAD staining. 7-AAD/Ki-67 staining was performed to discriminate between G0 and G1 phases. WT, n = 9; th3, n = 8. (C) Frequency of cycling HSCs expressed as a percentage of LRCs, revealing BrdU+ HSCs by in vivo BrdU pulse-chase assay. Chase fold-decrease, compared with respective pulse labeling is reported. WT pulse, n = 3; WT chase, n = 5; th3 pulse, n = 3; th3 chase, n = 3. (D-E) GSEA enrichment plots of the cell cycle–associated categories from the biological process branch of the GO database, showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list. Enrichment of th3 HSCs vs WT HSCs is reported. (F) Representative images of γH2AX+ foci in the nuclei of WT and th3 Lin− cells. Analysis of the frequency of cells accumulating DNA damage, shown as a percentage of γH2AX+ cells, is reported. WT, n = 3; th3, n = 3. Immunofluorescence analysis by anti-phospho-H2AX (Ser139) staining was performed. Cell nuclei were stained with Hoechst 33342. (G) GSEA enrichment plot of the cellular response to stress category from the Reactome Pathway database, showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list. Enrichment in th3 HSCs vs WT HSCs is reported. (H) GSEA enrichment plot showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list of key genes reported to be associated with HSC stemness. Enrichment in th3 HSCs vs WT HSCs is reported. (I) Methylcellulose CFU replating assay using sorted HSCs from WT and th3 mice. Absolute number of CFUs per plate is shown at different platings. WT, n = 6; th3, n = 6. *P < .05; **P < .01. NES, normalized enriched score.

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