PTEN expression in U87MG cells increases the formation of autophagosomes.

<p><b>A</b>) U87MG cells expressing WT-PTEN, C124S-PTEN and G129E-PTEN or mock-treated were incubated in high (H) or low (L) proteolysis media (see Materials and Methods) for 2 h in the presence of lysosomal inhibitors (100 µM leupeptin and 20 mM NH₄Cl). Extracts (75 µg protein) were analyzed by SDS-PAGE and immunoblot, with low and high exposure (exp.), using an antibody against LC3 and, as a loading control, an antibody that recognizes actin. A representative experiment is shown. The position of LC3-I and LC3-II bands are indicated on the left and molecular weight markers are indicated on the right. The histogram on the right shows the means ± SD of the densitometric analysis of the LC3-II/actin ratios from five different experiments. Stars indicate statistically significant differences from the corresponding (high or low proteolysis conditions) values in mock-treated cells at **<i>p</i><0.01 and ***<i>p</i><0.005. <b>B</b>) Representative fluorescent images of the indicated cell lines transitorily expressing EGFP-LC3 (at 48 h post-transfection) and incubated under high proteolysis conditions for 2 h. Quantification of autophagy by counting the number of EGFP-LC3 dots in the transfected cells (see Materials and Methods) is shown on the right. Stars indicate statistically significant differences from mock-treated cells at ***<i>p</i><0.005. Bar: 20 µm.