FIGURE 1.

Pterostilbene treatment elicits autophagy in primary dermal fibroblasts and in skeletal muscle of wild-type mice. (A) Western blot analysis for LC3B and LAMP1 in protein extracts of primary dermal fibroblasts from wild-type mice, treated with vehicle or with 15 μM Pt and incubated (+) or not (–) with chloroquine (CQ). β-actin and vinculin were used as loading controls. (B,C) Densitometric quantifications of LC3B-II vs. β-actin (B) and LAMP1 vs. vinculin (C), as determined by at least three independent western blot experiments as in (A). Data are shown as mean ± s.e.m. (n = 4–6, each condition; *P < 0.05; **P < 0.01). (D) Immunofluorescence confocal images for LAMP1 (red) and LC3B (green) on primary dermal fibroblast cultures from wild-type mice, treated for 2.5 h with 15 μM Pt or with vehicle. Nuclei were counterstained with Hoechst (blue). The dotted area is shown at higher magnifications on the right. Scale bar, 100 or 10 μm (magnifications). (E) Western blot analysis for LC3B in protein extracts of TA muscles from wild-type mice treated with a single oral gavage of vehicle or Pt (90.2 mg/kg body weight) and sacrificed 8 h after the treatment. Autophagic flux was assessed by i.p. injection of colchicine (CCH, +) or physiological solution (–). GAPDH was used as a loading control. (F) Densitometric quantification of LC3B-II vs. GAPDH, as determined by at least three independent western blot experiments as in (E). Data are shown as mean ± s.e.m. (n = 3–4, each condition; *P < 0.05). Veh, vehicle.