Fig. 4.

Rb2 activates autophagic flux in senescent HDFs. (A) Rb2 induces autophagic flux. Old HDFs transfected with the RFP-GFP-LC3 plasmid were treated with 20 μM of Rb2 for 24 h. The cells were analyzed with a confocal microscope. Quantification of yellow and red puncta (n = 10), ∗P < 0.05. (B) Rb2 decreased p62 protein level. Old HDFs treated with 10, 20 and 40 μM of Rb2 were harvested for immunoblot. p62 was detected with the specific antibody. The relative expression of p62 was shown in the graph, ∗P < 0.05. (C) Immunoblot analysis of autophagic flux. Old HDFs were treated with 20 μM of Rb2 in the presence or absence of 20 nM of BafA1. LC3 and p62 bands were normalized with β-actin and are shown in the graph, ∗P < 0.05. (D) Rb2 activated lysosomal function. Old HDF cells treated with 10 and 20 μM of Rb2 were stained with lysosome staining dye (LysoTracker green). The fluorescence intensity was measured using ImageJ software and the lysotracker intensity was normalized with Hoechst, ∗P < 0.05. (E) Rb2 increased formation of ultrastructure of autophagy. Red arrow: autophagosome, yellow arrow: autolysosome. The number of autophagosomes and autolysosomes per area was quantified (n = 10).