Figure 1: Pharmacological inhibition of autophagy restores Δ133p53α expression.

(a) Δ133p53α expression is induced by cycloheximide (CHX). MRC-5 fibroblasts (at population doubling levels (PDLs) 51) were treated with CHX (25 μg ml⁻¹) for indicated time periods and examined in immunoblot for Δ133p53α and full-length p53. β-actin was a loading control for normalization. The relative expression levels of Δ133p53α and full-length p53 are shown below the images. (b) Δ133p53α expression is induced by phosphoinositide 3-kinase inhibitors. MRC-5 fibroblasts (PDLs 51) were treated for 4 h with LY303511 (10 μM), LY294002 (10 μM), Wortmannin (50 nM) or dimethylsulphoxide alone (as control) and examined in immunoblot for Δ133p53α and β-actin expression. (c) Diminished Δ133p53α at replicative senescence is restored by treatment with bafilomycin A1. The immunoblot analyses were performed in early-passage (Y) and replicatively senescent (S) human fibroblast strains MRC-5 and WI-38. The examined PDLs were 30 (Y) and 60 (S) for MRC-5; and 30 (Y) and 54 (S) for WI-38. Bafilomycin A 1 treatment was at 100 nM for 4 h. The relative expression levels of Δ133p53α, full-length p53 and p62/SQSTM1 (normalized with β-actin) are shown. Anti-LC3B antibody detected LC3-I and LC3-II, which was used in quantitative analysis shown in d. (d) Replicative senescence is not associated with enhanced autophagy in general. LC3-II expression levels were normalized with β-actin (data in c) and are shown in the bar graphs as relative values to replicatively senescent cells without bafilomycin A1 (S/−; defined as 1). The LC3-II levels in replicatively senescent cells were higher than those in early-passage cells whether in the presence or absence of bafilomycin A1, which may suggest increased synthesis of autophagosomes at replicative senescence^7,37. However, the differences in LC3-II levels between the presence and absence of bafilomycin A1 were similar in early-passage and replicatively senescent cells: in MRC-5, 1.9 for Y (2.1−0.2) and 2.0 for S (3.0−1); in WI-38, 1.6 for Y (2.2−0.6) and 1.6 for S (2.6−1). These data indicate that early-passage and replicatively senescent cells have similar rates of autophagic flux, that is, the number of autophagosomes that are delivered to lysosomes for degradation^7,37.