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. 2014 Feb 7;28(8):1636–1646. doi: 10.1038/leu.2014.19

Figure 3.

Figure 3

MGCD0103 inhibits autophagy in primary CLL cells through activation of the PI3K/AKT/mTOR pathway and caspases. (a, b) The effect of MGCD0103 on the PI3K/AKT/mTOR pathway was assessed in primary CLL cells by immunoblotting through the analysis of the phosphorylation status of key regulators. (a) Time course experiments were performed with 3 μmol/l of MGCD0103 and (b) the effect of the 24-h treatment was analyzed using two concentrations of MGCD0103 (0.5 and 3 μmol/l, as indicated). Relative protein quantification is available in Supplementary Table 2. Blots from three independent experiments are shown. (c, d) MGCD0103 decreases the autophagic flux in primary CLL cells in a caspase-dependent manner. Cells were incubated for 24 h alone or in the presence of MGCD0103 (3 μmol/l) or VPA (4 mmol/l), with or without chloroquine (CQ; 20 μmol/l), bafilomycin A1 (Baf; 20 nmol/l), 3-MA (1 mmol/l), bortezomib (Bort; 2 nmol/l) or Q-VD-OPh (Q-VD; 10 μmol/l). Caspase and proteasome inhibitors were added to PBMCs 1 h before and autophagy inhibitors 1 h after MGCD0103 or VPA. Two representative blots (c and d) from five independent experiments are shown. The levels of indicated proteins were analyzed by immunoblotting, using ACTB as a loading control. The expression levels of proteins in treated cells relative to the untreated control and normalized to ACTB levels are indicated.