Figure 4. Ablation of the Catalytic Domain of SIRT1 Results in Increased Global H4K16ac and Precocious Activation/Differentiation of SCs.

(A) Skeletal muscle from WT mice demonstrated the presence of the SIRT1 floxed allele (fl, top panel) and detectable levels of SIRT1 protein (arrow, bottom panel), while SIRT1^mKO muscle contained the SIRT1^Δex4 allele (Δ, top panel) and ablation of SIRT1 protein. A small level of SIRT1^Δex4 protein was detectable in the skeletal muscle of SIRT1^mKO mice (arrowhead, bottom panel). (B, C) In SIRT1^mKO mice, quiescent SCs (identified as Pax7⁺) exhibited a two-fold increase in global H4K16ac, compared to WT mice, as determined via relative fluorescence (RFU) in SCs labelled for H4K16ac (n=2 mice, >50 fibers/timepoint). White scale bar indicates 50μm, inset is magnified by a magnitude of four. Results are presented as box-and-whisker plots (5–95 percentiles), with a significant difference indicated when the median ± 95% CI does not overlap. (D, E) MyoD and Pax7 staining (n=2 mice, >50 fibers/timepoint) of fiber-associated SIRT1^mKO SCs. Data is presented as mean ± SEM, *p < 0.05. (F) DIC images of SCs isolated from WT and SIRT1^mKO mice during proliferation in growth media (GM, 48hrs), or early differentiation in differentiation media (24hrs DM). Note the overt spindle-like, elongated morphology of SCs from SIRT1^mKO mice indicating premature differentiation.