Figure 1
Figure 1. DNA damage and gene expression changes in response to telomere shortening accumulate in quiescent HSCs but not in hematopoietic progenitors and myeloid cells. (A) The histogram shows the number of genes that are differentially expressed in HSCs, MPPs, and myeloid cells isolated from 12-month-old G3mTerc−/− compared with age-matched mTerc+/+ mice. Fifty to 200 freshly isolated cells were used for the analysis of gene expression profiles (n = 6-7 mice per group). Several DNA damage–related or apoptosis-related genes were differentially expressed in HSCs between G3mTerc−/− compared with age-matched mTerc+/+ mice, including Polq (polymerase, θ), Fancc (Fanconi anemia, complementation group C), Rb1 (Retinoblastoma 1), Ccdc14 (coiled-coil domain containing 14), Bmf (Bcl2 modifying factor), Aatf (apoptosis antagonizing transcription factor), Hspa12b (heat shock protein A12B), Anxa9 (Annexin 9), Ddx10 (Dead box 10), Dact2 (Dapper homolog 2), Rbm10 (RNA binding motif protein 10), Map3k12 (mitogen-activated protein kinase kinase kinase 12), and Socs6 (suppressor of cytokine signaling 6). (B-G) To analyze DNA damage, HSCs, MPPs, and myeloid cells from 12-month-old G3mTerc−/− mice and mTerc+/+ mice were freshly isolated for DNA damage quantification assay including γH2AX staining and alkaline comet assay. (B-D) The histograms show the percentage of cell nuclei staining positive for the indicated numbers of γH2AX foci in HSCs (B), MPPs (C), and myeloid cells (D) (100 nuclei were counted per cell type and group). (E-G) DNA fragmentation was analyzed using the alkaline comet assay. Fragmented DNA is visualized as a tail moving out of the gel-embedded nuclei. More than 500 HSCs, MPPs, and myeloid cells from G3mTerc−/− and mTerc+/+ mice were scored by alkaline comet assay. The histogram shows the distribution of tail DNA percentage of HSCs (E), MPPs (F), and myeloid cells (G) from 12-month-old G3mTerc−/− and age-matched mTerc+/+ mice (n > 500 nuclei per group). (H) Representative images of HSCs from 12-month-old G3mTerc−/− and age-matched mTerc+/+ mice analyzed by the alkaline comet assay.

DNA damage and gene expression changes in response to telomere shortening accumulate in quiescent HSCs but not in hematopoietic progenitors and myeloid cells. (A) The histogram shows the number of genes that are differentially expressed in HSCs, MPPs, and myeloid cells isolated from 12-month-old G3mTerc−/− compared with age-matched mTerc+/+ mice. Fifty to 200 freshly isolated cells were used for the analysis of gene expression profiles (n = 6-7 mice per group). Several DNA damage–related or apoptosis-related genes were differentially expressed in HSCs between G3mTerc−/− compared with age-matched mTerc+/+ mice, including Polq (polymerase, θ), Fancc (Fanconi anemia, complementation group C), Rb1 (Retinoblastoma 1), Ccdc14 (coiled-coil domain containing 14), Bmf (Bcl2 modifying factor), Aatf (apoptosis antagonizing transcription factor), Hspa12b (heat shock protein A12B), Anxa9 (Annexin 9), Ddx10 (Dead box 10), Dact2 (Dapper homolog 2), Rbm10 (RNA binding motif protein 10), Map3k12 (mitogen-activated protein kinase kinase kinase 12), and Socs6 (suppressor of cytokine signaling 6). (B-G) To analyze DNA damage, HSCs, MPPs, and myeloid cells from 12-month-old G3mTerc−/− mice and mTerc+/+ mice were freshly isolated for DNA damage quantification assay including γH2AX staining and alkaline comet assay. (B-D) The histograms show the percentage of cell nuclei staining positive for the indicated numbers of γH2AX foci in HSCs (B), MPPs (C), and myeloid cells (D) (100 nuclei were counted per cell type and group). (E-G) DNA fragmentation was analyzed using the alkaline comet assay. Fragmented DNA is visualized as a tail moving out of the gel-embedded nuclei. More than 500 HSCs, MPPs, and myeloid cells from G3mTerc−/− and mTerc+/+ mice were scored by alkaline comet assay. The histogram shows the distribution of tail DNA percentage of HSCs (E), MPPs (F), and myeloid cells (G) from 12-month-old G3mTerc−/− and age-matched mTerc+/+ mice (n > 500 nuclei per group). (H) Representative images of HSCs from 12-month-old G3mTerc−/− and age-matched mTerc+/+ mice analyzed by the alkaline comet assay.

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