Inhibition of the ubiquitin-proteasome pathway and activation of autophagy by PTEN in U87MG cells are independent of mTOR.

<p>U87MG cells expressing WT-PTEN or C124S-PTEN (<b>A</b>) or WT-PTEN or G129E-PTEN and mock-treated (<b>B</b>) were incubated for 18 h with doxycycline and in the last 2 h the following inhibitors were added as indicated: rapamycin (RAP, 200 mM, mTOR inhibitor), KT5720 (25 µM, PKA inhibitor), PD98059 (10 µM, ERK1/2 inhibitor) and SB203580 (10 µM, p38 inhibitor). Before collecting the cells, proteasome (50 µM MG132, <b>A</b>) and lysosomal (100 µM leupeptin and 20 mM NH₄Cl, B) inhibitors were also added for 1 h. Total lysates were analyzed by SDS-PAGE and Western blot with antibodies that recognize ubiquitinated proteins (FK1) (<b>A</b>), LC3 (<b>B</b>) and, as a loading control actin. Molecular weight markers are indicated on the right and in <b>B</b> the position of LC3-I, and LC3-II bands are also shown. The histograms on the right show means ± SD of the densitometric measurements from three different experiments and are expressed as amounts of ubiquitinated proteins (ubiq. prot.) normalized to the levels of actin (<b>A</b>) or as LC3II/actin ratios (<b>B</b>). Stars indicate statistically significant differences from the values without the corresponding inhibitor treatment at **<i>p</i><0.01 and ***<i>p</i><0.005.