Figure 3.
Dcaf8 knockout leads to cellular senescence and DNA damage in HSCs. (A-D) Representative images of (A) P16 and (C) P21 indicated by immunofluorescence, with quantification of (B) P16 (n = 64 cells for WT; n = 67 cells for Dcaf8−/−, pooled from 3 mice) and (D) P21 (n = 39 cells for WT; n = 36 cells for Dcaf8−/−, pooled from 3 mice) by MFI in purified LT-HSCs from young WT and Dcaf8−/− mice. Scale bar, 5 μm. (E) RT-qPCR of p16, p21, and p53 in purified LT-HSCs from young WT and Dcaf8−/− mice (n = 4 technical replicates, pooled from 3 mice). (F-G) Representative images of (F) senescence-associated β-galactosidase (SA-β-gal) staining and (G) quantification in BM cells from young WT, Dcaf8−/− mice, and aged (20 months old) WT controls (n = 3 independent experiments, each pooled from 3 mice). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (H-I) Representative images of (H) phosphorylated H2A histone family member X (γ-H2AX) indicated by immunofluorescence and (I) quantification of γ-H2AX foci in purified LT-HSCs from young WT and Dcaf8−/− mice (n = 34 cells for WT; n = 40 cells for Dcaf8−/−, pooled from 3 mice). Scale bar, 5 μm. Statistical analysis used 2-tailed Mann-Whitney U test. (J) Representative flow cytometric plots and (K) analysis of γ-H2AX expression of LT-HSCs from young WT and Dcaf8−/− mice (n = 4 mice). (L) Representative alkaline comets and quantification by (M) percent of DNA in tail and (N) olive tail moment of purified LT-HSCs from young WT (n = 65 cells, pooled from 3 mice), Dcaf8−/− (n = 79 cells, pooled from 3 mice) mice, and aged (20 months old) WT controls (n = 76 cells, pooled from 3 mice). Statistical analysis used Kruskal-Wallis test followed by the Dunn multiple comparisons test. (O) Analysis of reactive oxygen species levels stained with DCFH-DA by flow cytometry of LT-HSCs from young WT (n = 3 mice), Dcaf8−/− (n = 4 mice) mice, and aged (20 months old) WT controls (n = 4 mice). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. For panels I, M, and N, data are presented as mean ± SEM; other data are presented as mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. ns, no statistical significance; ROS, reactive oxygen species.

Dcaf8 knockout leads to cellular senescence and DNA damage in HSCs. (A-D) Representative images of (A) P16 and (C) P21 indicated by immunofluorescence, with quantification of (B) P16 (n = 64 cells for WT; n = 67 cells for Dcaf8−/−, pooled from 3 mice) and (D) P21 (n = 39 cells for WT; n = 36 cells for Dcaf8−/−, pooled from 3 mice) by MFI in purified LT-HSCs from young WT and Dcaf8−/− mice. Scale bar, 5 μm. (E) RT-qPCR of p16, p21, and p53 in purified LT-HSCs from young WT and Dcaf8−/− mice (n = 4 technical replicates, pooled from 3 mice). (F-G) Representative images of (F) senescence-associated β-galactosidase (SA-β-gal) staining and (G) quantification in BM cells from young WT, Dcaf8−/− mice, and aged (20 months old) WT controls (n = 3 independent experiments, each pooled from 3 mice). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. (H-I) Representative images of (H) phosphorylated H2A histone family member X (γ-H2AX) indicated by immunofluorescence and (I) quantification of γ-H2AX foci in purified LT-HSCs from young WT and Dcaf8−/− mice (n = 34 cells for WT; n = 40 cells for Dcaf8−/−, pooled from 3 mice). Scale bar, 5 μm. Statistical analysis used 2-tailed Mann-Whitney U test. (J) Representative flow cytometric plots and (K) analysis of γ-H2AX expression of LT-HSCs from young WT and Dcaf8−/− mice (n = 4 mice). (L) Representative alkaline comets and quantification by (M) percent of DNA in tail and (N) olive tail moment of purified LT-HSCs from young WT (n = 65 cells, pooled from 3 mice), Dcaf8−/− (n = 79 cells, pooled from 3 mice) mice, and aged (20 months old) WT controls (n = 76 cells, pooled from 3 mice). Statistical analysis used Kruskal-Wallis test followed by the Dunn multiple comparisons test. (O) Analysis of reactive oxygen species levels stained with DCFH-DA by flow cytometry of LT-HSCs from young WT (n = 3 mice), Dcaf8−/− (n = 4 mice) mice, and aged (20 months old) WT controls (n = 4 mice). Statistical analysis used 1-way ANOVA followed by the Tukey multiple comparisons test. For panels I, M, and N, data are presented as mean ± SEM; other data are presented as mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by unpaired 2-tailed t tests unless otherwise specified. ns, no statistical significance; ROS, reactive oxygen species.

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