Figure 2: Muscle regeneration in type I diabetes by RSV in a Sirt1-independent manner. (A) Western blotting of myoblasts transfected with lentiviral particles delivering Sirt1 shRNA shows effective knock down of Sirt1 protein expression; n=3 separate cultures. (B) Satellite fiber-associated cells were prepared from TA and Gastroc muscle of control, STZ-treated mice (STZ), insulin-treated STZ mice (STZ/INS) and RSV-treated STZ mice (STZ/RSV) at 3 DPI with or withoutSirt1 shRNA infection, cultured in growth medium for 24 h, pulsed with BrdU for 2 hours and then switched to differentiation medium for 48 h. Formation of de-novo eMyHC+myotubes (Red) and BrdU+ nuclei (Green) were assayed by immunofluorescence. Hoechst (Blue) labels all nuclei. Scale bar, 100 μm. (C) The percent of nuclei in eMyHC+ myotubes to total nuclei (black bars), the percent of eMyHC+ myotubes with more than two nuclei to total nuclei (white bars), and the percent of BrdU+ nuclei in myotubes to total nuclei (gray bars) were quantified. n=3 with more than three independent experiments. (D) Satellite cells were prepared from TA and Gastroc muscle of control, STZ-treated mice (STZ), inuslin-treated STZ mice (STZ/INS) and RSV-treated STZ mice (STZ/RSV) at 3 DPI with EX-527 administration, cultured in growth medium for 24 h and then switched to differentiation medium for 48 h. Formation of de-novo eMyHC+ myotubes (Red) and BrdU+ nuclei (Green) were assayed by immunofluorescence. Hoechst (Blue) labels all nuclei. Scale bar, 100 μm. (E) The percent of nuclei in eMyHC+ myotubes to total nuclei (black bars), the percent of eMyHC+myotubes with more than two nuclei to total nuclei (white bars), and the percent of BrdU+ nuclei in myotubes to total nuclei (gray bars) were quantified. n=3 with more than three independent experiments.