Fig 2: Analysis of the sirtuin proteins activity and expression. Immunoblotting analysis of SIRT1 and SIRT3 was performed in the hearts (A,B). The columns represent the average ratios of relative protein expression in the control and irradiated samples after background correction and normalisation to VDAC expression (one-way ANOVA and Tukey test for multiple comparisons t-test; * adjusted p value < 0.05; n = 3) (B). The activity of total SIRTs was compared between irradiated and control samples (one-way ANOVA followed by Tukey’s multiple comparisons test; * adjusted p value < 0.05; n = 5) (C). The error bars represent the standard error of the mean (±SEM).

Fig 3: Sirt3 controls the acetylation status of FoxO1A-B. Immunoblot analysis of FoxO1 and ac-FoxO1 was performed in sham and AngII-treated WT and Sirt3-KO murine hearts. Tubulin expression was used as loading control. Bar graph represents quantification of ac-FoxO1 levels measured by densitometry analysis. (n=5) C-D. Immunoblot analysis of autophagy biomarkers and Sirt3 in the control and siRNA-FoxO1 groups. GAPDH expression was used as loading control. Bar graph represents quantification of LC3-II levels measured by densitometry analysis. (n=5) E. Cellular extracts were immunoprecipitated with Sirt3 antibody and analyzed with anti-FoxO1. F. Cellular extracts were immunoprecipitated with FoxO1 antibody and analyzed with anti-Sirt3. The data are presented as the means ± SEM of three independent experiments.*P<0.05, **P<0.01.

Fig 4: Sirt3 expression depends on Atg7 in the mouse bone marrow HSC‐enriched hematopoietic cells. (a) The volcano map of differentially expressed genes in Atg7−/− HSC‐enriched hematopoietic cells as compared to Atg7+/+ HSC‐enriched cells. A total of 1062 genes were significantly upregulated, while 789 genes were downregulated in Atg7‐depleted HSC‐enriched cells. (b) RNA sequencing reveals Sirt3 dependency on Atg7. Left, the heatmap of Sirtuin family between Atg7+/+ and Atg7−/− HSC‐enriched cells. The result shows that only Sirt3 expression is reduced in the HSPCs of all of the three Atg7−/− mice. Middle, the reduction levels of the expression of seven Sirtuin family members in the HSC‐enriched cells due to Atg7 deletion. The result shows that only Sirt3 expression is significantly reduced in the Atg7−/− HSC‐enriched cells. Right, the relative expression levels of Sirtuin family members in wild‐type HSC‐enriched cells. The result shows that Sirt3 is the highest expressed member in Sirtuin family in the HSC‐enriched cells of the wild‐type mice. (c) Measurement of expression levels of the Sirtuin family members by quantitative PCR in the Atg7+/+ and Atg7−/− HSC‐enriched cells. (d) Measurement of Sirt3 pre‐mRNA by quantitative PCR. pre‐mRNA expression was normalized to gapdh level. (e) Flow cytometric examination of Sirt3 protein expression in HSC‐enriched cells, hematopoietic progenitor cells, and terminally differentiated hematopoietic cells of wild‐type and Atg7‐deleted mice aged 10 weeks. Left, representative data of Sirt3 protein expression levels in HSC‐enriched cells (LSK), HPCs (Lin−), and terminally differentiated hematopoietic cells (Lin+) by flow cytometry. Right, statistic data for comparison of Sirt3 expression levels. (f) Measurement of expression of Sirt3, Atg7, and LC3 proteins by Western blotting in Atg7+/+ and Atg7−/− bone marrow mononuclear cells (MNCs) and hematopoietic progenitor cells (Lin− sorted) from 10‐week‐old mice. Gapdh serves as a loading control. (g) Confocal detection of Sirt3 protein in the Atg7+/+ and Atg7−/− HSC‐enriched cells. Representative images were taken from bone marrow HSC‐enriched cells of mice aged at 2 weeks and 10 weeks. (h) Quantitative PCR measurement of Sirt3 expression levels in the HSC‐enriched cells from aging wild‐type mice. (i) Time course comparison on Sirt3 expression by quantitative PCR in HSC‐enriched cells of wild‐type and Atg7‐deleted mice

Fig 5: SIRT3 inhibition improves response to venetoclax. (A) Colony-forming ability of 4 primary acute myeloid leukemia (AML) specimens treated with 10 µM YC8-02, 100 nM of venetoclax or a combination of the two, treated for 72 hours prior to performing colony-forming unit (CFU) assay. Statistical significance was determined using an ordinary one-way ANOVA. (B) Engraftment of AML 7 treated with with 10 µM YC8-02, 100 nM of venetoclax, or a combination of the two for 24 hours prior to engraftment. Each point represents a single mouse. Statistical significance was determined using ordinary one-way ANOVA. All error bars represent standard deviation. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001; ns: not significant.