SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation

Figure 4

SIRT3 is required for MSC differentiation. (a) Passage 3 MSCs were transduced with retrovirus expressing shRNA against SIRT3 or scrambled control. Two different SIRT3 shRNA sequences were used. Western blotting was used to demonstrate efficient depletion of SIRT3. (b) To assess senescence, β-galactosidase activity assay was performed. Bars represent means ± SEM for the 3 MSC strains. from two-sided t-tests comparing shSIRT3-1 and shSIRT3-2 to both untreated and shScrambled conditions. (c) To assess ROS, MSCs were stained with DHE, which was detected by flow cytometry. Bars represent average DHE values (median channel fluorescence from FL3) ± SEM from the 3 MSC strains. “Untreated” refers to wild-type MSCs that were not virally transduced. from two-sided t-tests comparing shSIRT3-1 and shSIRT3-2 to both untreated and shScrambled conditions. (d) MSCs were differentiated for 21 days with appropriate adipogenic or osteogenic induction media. Adipocytes were stained with oil red O, and osteoblasts were stained with alizarin red S. Representative images are shown. Wild-type MSCs not treated with differentiation media but still stained with either oil red O or alizarin red S served as controls. Scale . (e) Isopropanol was used to elute the oil red O and alizarin red S, and absorbance was read at 500 nm or 520 nm, respectively. Bars represent means ± SEM for 3 MSC strains. from two-sided t-tests comparing shSIRT3-1 and shSIRT3-2 to shScrambled.

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