Figure 1.
Aging-related decreased expression of DCAF8 leads to increased number of HSCs. (A) Reverse transcription qPCR (RT-qPCR) of Dcaf8 in various murine HSPCs, including LT-HSC (Lin−Sca-1+c-Kit+CD150+CD48−), ST-HSC (Lin−Sca-1+c-Kit+CD150−CD48−), MPP (Lin−Sca-1+c-Kit+CD48+), CMP (Lin−Sca-1−c-Kit+CD34+CD16/32−), CLP (Lin−Sca-1loc-KitloCD127+), and Lin− cells (n = 3 technical replicates, pooled from 3 mice). (B) Immunoblot of DCAF8 in LK cells at different mouse ages. (C) The relative intensity of DCAF8 immunoblot in LK cells, quantified and normalized to β-actin (n = 3 independent experiments). (D) Immunoblot of DOCK11 in LT-HSCs at different mouse ages. (E) The relative intensity of DOCK11 immunoblot in LT-HSCs, quantified and normalized to β-actin (n = 3 independent experiments). (F) Representative flow cytometric plots for HSPCs in the BM of 8-week-old WT and Dcaf8−/− mice. (G-H) Frequency of LT-HSC, ST-HSC, MPP2 (Lin−Sca-1+c-Kit+CD150+CD48+), and MPP3 (Lin−Sca-1+c-Kit+CD150−CD48+) represented as percentages of (G) total BM cells (n = 6 mice) and (H) LSK (Lin−Sca-1+c-Kit+) cells (n = 6 mice) in 8-week-old WT and Dcaf8−/− mice. (I) Surface expression of CD150 of LT-HSCs in WT and Dcaf8−/− mice (n = 6 mice), expressed as mean fluorescence intensity (MFI) analyzed by flow cytometry. (J) Frequency of LT-HSC divided into CD150high and CD150low subgroups represented as percentages of total BM cells in 8-week-old WT and Dcaf8−/− mice (n = 6 mice). (K) Frequency of LT-HSC represented as percentages of LSK cells at different ages in WT and Dcaf8−/− groups (n = 4, 4, 5, 3, and 4 mice for WT; and 5, 4, 4, 3, and 4 mice for Dcaf8−/−). Statistical analysis used 2-way analysis of variance (ANOVA) followed by the Tukey multiple comparisons test. (L) Number of LT-HSC in BM at different ages in WT and Dcaf8−/− groups (n = 4 mice). (M-N) Frequency of (M) myeloid (CD11b+) and (N) B cells (CD19+) in the PB at different ages in WT and Dcaf8−/− mice (n = 12, 14, 9, 6, and 5 mice for WT; and 11, 10, 7, 5, and 4 mice for Dcaf8−/−). Statistical analysis used 2-way ANOVA followed by the Tukey multiple comparisons test. Data are presented as mean ± standard deviation (SD). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. CLP, common lymphoid progenitors; CMP, common myeloid progenitors; LK, Lin−c-Kit+; MPP, multipotent progenitors; ns, no statistical significance; ST-HSC, short-term HSC.

Aging-related decreased expression of DCAF8 leads to increased number of HSCs. (A) Reverse transcription qPCR (RT-qPCR) of Dcaf8 in various murine HSPCs, including LT-HSC (Lin−Sca-1+c-Kit+CD150+CD48−), ST-HSC (Lin−Sca-1+c-Kit+CD150−CD48−), MPP (Lin−Sca-1+c-Kit+CD48+), CMP (Lin−Sca-1−c-Kit+CD34+CD16/32−), CLP (Lin−Sca-1loc-KitloCD127+), and Lin− cells (n = 3 technical replicates, pooled from 3 mice). (B) Immunoblot of DCAF8 in LK cells at different mouse ages. (C) The relative intensity of DCAF8 immunoblot in LK cells, quantified and normalized to β-actin (n = 3 independent experiments). (D) Immunoblot of DOCK11 in LT-HSCs at different mouse ages. (E) The relative intensity of DOCK11 immunoblot in LT-HSCs, quantified and normalized to β-actin (n = 3 independent experiments). (F) Representative flow cytometric plots for HSPCs in the BM of 8-week-old WT and Dcaf8−/− mice. (G-H) Frequency of LT-HSC, ST-HSC, MPP2 (Lin−Sca-1+c-Kit+CD150+CD48+), and MPP3 (Lin−Sca-1+c-Kit+CD150−CD48+) represented as percentages of (G) total BM cells (n = 6 mice) and (H) LSK (Lin−Sca-1+c-Kit+) cells (n = 6 mice) in 8-week-old WT and Dcaf8−/− mice. (I) Surface expression of CD150 of LT-HSCs in WT and Dcaf8−/− mice (n = 6 mice), expressed as mean fluorescence intensity (MFI) analyzed by flow cytometry. (J) Frequency of LT-HSC divided into CD150high and CD150low subgroups represented as percentages of total BM cells in 8-week-old WT and Dcaf8−/− mice (n = 6 mice). (K) Frequency of LT-HSC represented as percentages of LSK cells at different ages in WT and Dcaf8−/− groups (n = 4, 4, 5, 3, and 4 mice for WT; and 5, 4, 4, 3, and 4 mice for Dcaf8−/−). Statistical analysis used 2-way analysis of variance (ANOVA) followed by the Tukey multiple comparisons test. (L) Number of LT-HSC in BM at different ages in WT and Dcaf8−/− groups (n = 4 mice). (M-N) Frequency of (M) myeloid (CD11b+) and (N) B cells (CD19+) in the PB at different ages in WT and Dcaf8−/− mice (n = 12, 14, 9, 6, and 5 mice for WT; and 11, 10, 7, 5, and 4 mice for Dcaf8−/−). Statistical analysis used 2-way ANOVA followed by the Tukey multiple comparisons test. Data are presented as mean ± standard deviation (SD). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. CLP, common lymphoid progenitors; CMP, common myeloid progenitors; LK, Lin−c-Kit+; MPP, multipotent progenitors; ns, no statistical significance; ST-HSC, short-term HSC.

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