Figure 2

MGCD0103 decreases the autophagic flux in primary CLL cells. (a, b) The effect of MGCD0103 (3 μmol/l) on autophagy was assessed by immunoblotting in the presence of chloroquine in time course experiments. ACTB was used as a loading control. Two representative blots (a and b) from four independent experiments are shown. The expression levels of proteins in treated cells relative to the corresponding untreated control and normalized to ACTB levels are indicated. (c, d) Autophagy was assessed by flow cytometry using the Cyto-ID green autophagy dye. For the left panel in (c), single-cell lymphocyte population was gated in R1 (live and apoptotic cells), whereas apoptotic cells were gated in R2 on the basis of cell shrinkage (decreased in forward scatter (FSC)) and increase in granularity (increase in side scatter (SSC)). Shown on the right panel of (c) are the distributions of the dye green fluorescence (channel FL1) within each R1 population. Dot plots and Cyto-ID green fluorescence intensity from a representative experiment are shown in (c). (d) Results are represented by histograms as mean Cyto-ID green fluorescence of treated cells relative to control cells (dark bars). The percentage of apoptotic cells represents the percentage of cells in R2 (bright bars). The mean values±s.d. (n=8) are shown. Values are compared with the corresponding control value. *P<0.05 and **P<0.01.