FIGURE 8.

DAXX K/D increases autophagic flux in cultured PCa cells PC3. A, PC3 cells, control (WT) and DAXX K/D (K/D), transiently transfected with GFP-LC3 construct for 24 h, were cultured under normal (Basal) conditions as described under “Experimental Procedures.” The GFP-LC3 puncta were visualized using confocal microscopy (top two panels), and their numbers were assessed using IMARIS (bottom panel). At least 15 cells were analyzed. The number of LC3 puncta was increased in DAXX K/D, indicating higher basal autophagy in these cells. B, PC3 cells, control (WT) and DAXX K/D (K/D), were exposed to a time course of BafA (an inhibitor of autophagic flux) treatment (0–4 h). Cell lysates were subjected to immunoblotting with the indicated antibodies. DAXX and β-actin blots are included as controls. p62 levels were increased in WT cells upon autophagy inhibition, suggesting decreased autophagy. Similarly, whereas p62 levels were low in DAXX K/D under basal conditions (0 h), they were increased at later time points of autophagy inhibition but remained statistically lower than the WT group (bottom), pointing to increased overall autophagic flux in DAXX K/D compared with WT cells. ImageJ was used to quantify the blots, and GraphPad Prism version 5.0 was employed to construct the graph and perform statistical analyses. Error bars, S.E.