Figure 5.

Cellular Location of p27^Kip1 Regulates MuSC Function in Part through the Induction of Autophagy

(A) Upper: p27^Kip1 protein expression in both cytoplasmic and nuclear compartments of freshly sorted MuSCs across age groups. Lower: quantification of percentage of cytoplasmic and nuclear expression of p27^Kip1 (N = 3 independent experiments).

(B) Upper: p27^Kip1 protein expression in both cytoplasmic and nuclear compartments of activated MuSCs (48 hr in culture) across age groups. Lower: quantification of percentage of cytoplasmic and nuclear expression of p27^Kip1 (N = 3 independent experiments).

(C) p27^Kip1 phosphorylation status-mediated cellular location and autophagy. Left: immunofluorescence staining of LC3B and p27^Kip1 expression of young MuSCs overexpressing GPF-labeled WT p27^Kip1, p27^Kip1(198A), or p27^Kip1(198D). All cells were treated with chloroquine for the final 12 hr of culture. LC3B, red; p27^Kip1, green; DAPI, blue. Scale bar, 5 μm in (C). Right: quantification of relative change in LC3B puncta across treatment groups (N = 3 independent experiments).

(D) LC3B isoform protein expression in young MuSCs overexpressing WT p27^Kip1, p27^Kip1(198A), or p27^Kip1(198D).

(E) Upper: EdU incorporation in control or p27^Kip1(198A) overexpressing young MuSCs. Lower: quantification of percentage of EdU(+) MuSCs in each group (N = 3 independent experiments).

(F) Upper: EdU incorporation in control, p27^Kip1(198A) overexpressing or p27^Kip1(198D) overexpressing geriatric MuSCs with or without Atg5 knockdown. Lower: quantification of percentage of EdU(+) MuSCs in each group (N = 3 independent experiments).

^∗ Signifies difference from young or control cells; † signifies difference from old cells (p < 0.05). Scale bars, 100 μm in (E) and (F). Data are presented as means ± SE.