Figure 5. Loss of Sirt1 causes increased... | Rockefeller University Press

Figure 5.

$Figure 5. Loss of Sirt1 causes increased sensitivity to DNA damage. (A) FACS analysis of c-Kit and Sca-1 expression in Sirt1-E4fl/fl (WT) or Sirt1-E4fl/fl, ERT2-Cre (SIRT1Δ/Δ) BM cells cultured for 10 d in SCF, TPO, FLT3L, and IL-6 (4F). One of at least three representative experiments is shown. (B) BM cultures from SIRT1Δ/Δ mice and WT controls maintained in SCF alone or 4F were analyzed in triplicate for the fraction of γ-H2AX+ cells after 11 d in culture. A representative staining is shown. Error bars represent SD. (C) SIRT1Δ/Δ, WT, and ERT2-Cre control BM cultured in the presence of 4F for 11 d was subjected to IR (8 Gy) or left untreated (0 Gy) and analyzed 6 h thereafter for p53 expression and p53-K379 acetylation. GAPDH served as loading control. One of three representative experiments is shown. (D) FACS analysis of Annexin V+ cells in Sirt1-E4fl/fl, vav-iCre (SIRT1Δ/Δ) or vav-iCre (Cre) control BM after 11 d of 4F culture. Cultures were left untreated or exposed to irradiation (2 Gy or 9 Gy) and analyzed 6 h thereafter. Samples were analyzed in triplicate for the total fraction of Annexin V+ cells. A representative staining is shown. Error bars represent SD. (E) FACS analysis of Annexin V+ cells cultured for 11 d in the presence of SCF alone or 4F. Analysis was performed as in D. Error bars represent SD. (F) Metaphase analysis from Sirt1-E4fl/fl (WT) and Sirt1-E4fl/fl, ERT2-Cre (SIRT1Δ/Δ) 4F BM cultures. Sirt1 was deleted in vivo before culture. The frequency of total metaphases containing DNA aberrations is shown. DAPI images depict examples of individual aberrations. Bar, 10 µm. See Table 1 for details. Mean and SD are based on BM cultures from three animals per group. 50 metaphases were analyzed per sample. (G) Frequency of γ-H2AX+ LSK and Lin−c-Kit−Sca-1− non-HSPC cells in vivo from mice in Fig. 2 B. Recipients of Sirt1-deficient (SIRT1Δ/Δ, n = 5) or control (WT, n = 6) BM were analyzed 20 wk after transfer. Diamonds represent individual recipient mice. Horizontal bars represent the mean of all data points. (H) Sirt1-E4fl/fl, vav-iCre mice (SIRT1Δ/Δ, n = 10) and age-matched vav-iCre controls (Cre, n = 4) were exposed to sublethal IR (9 Gy) and survival was monitored over 1 mo. P = 0.045 (Wilcoxon Test); P = 0.049 (Log-rank). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; NS, not significant.$

Loss of Sirt1 causes increased sensitivity to DNA damage. (A) FACS analysis of c-Kit and Sca-1 expression in Sirt1-E4^fl/fl (WT) or Sirt1-E4^fl/fl, ERT2-Cre (SIRT1^Δ/Δ) BM cells cultured for 10 d in SCF, TPO, FLT3L, and IL-6 (4F). One of at least three representative experiments is shown. (B) BM cultures from SIRT1^Δ/Δ mice and WT controls maintained in SCF alone or 4F were analyzed in triplicate for the fraction of γ-H2AX⁺ cells after 11 d in culture. A representative staining is shown. Error bars represent SD. (C) SIRT1^Δ/Δ, WT, and ERT2-Cre control BM cultured in the presence of 4F for 11 d was subjected to IR (8 Gy) or left untreated (0 Gy) and analyzed 6 h thereafter for p53 expression and p53-K379 acetylation. GAPDH served as loading control. One of three representative experiments is shown. (D) FACS analysis of Annexin V⁺ cells in Sirt1-E4^fl/fl, vav-iCre (SIRT1^Δ/Δ) or vav-iCre (Cre) control BM after 11 d of 4F culture. Cultures were left untreated or exposed to irradiation (2 Gy or 9 Gy) and analyzed 6 h thereafter. Samples were analyzed in triplicate for the total fraction of Annexin V⁺ cells. A representative staining is shown. Error bars represent SD. (E) FACS analysis of Annexin V⁺ cells cultured for 11 d in the presence of SCF alone or 4F. Analysis was performed as in D. Error bars represent SD. (F) Metaphase analysis from Sirt1-E4^fl/fl (WT) and Sirt1-E4^fl/fl, ERT2-Cre (SIRT1^Δ/Δ) 4F BM cultures. Sirt1 was deleted in vivo before culture. The frequency of total metaphases containing DNA aberrations is shown. DAPI images depict examples of individual aberrations. Bar, 10 µm. See Table 1 for details. Mean and SD are based on BM cultures from three animals per group. 50 metaphases were analyzed per sample. (G) Frequency of γ-H2AX⁺ LSK and Lin⁻c-Kit⁻Sca-1⁻ non-HSPC cells in vivo from mice in Fig. 2 B. Recipients of Sirt1-deficient (SIRT1^Δ/Δ, n = 5) or control (WT, n = 6) BM were analyzed 20 wk after transfer. Diamonds represent individual recipient mice. Horizontal bars represent the mean of all data points. (H) Sirt1-E4^fl/fl, vav-iCre mice (SIRT1^Δ/Δ, n = 10) and age-matched vav-iCre controls (Cre, n = 4) were exposed to sublethal IR (9 Gy) and survival was monitored over 1 mo. P = 0.045 (Wilcoxon Test); P = 0.049 (Log-rank). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; NS, not significant.

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