Figure 4.

The effects of lysosomal enzyme inhibitors and 3-MA on altered localization and expression pattern of LC3 protein induced by PML-RARα. (A and B) U₂OS cells were transiently co-transfected with GFP-LC3 and DsRed-PML-RARα (bottom parts) or DsRed (upper parts) for 24 h, followed by treatment with or without 3-MA (10 mM) and pepstatin A (10 µg/ml) plus E64d (10 µg/ml) for an additional 4 h. Then the cells were observed by confocal microscopy. The representative images for each treatment are shown (A). Quantification data of the percentage of GFP-LC3 puncta-positive cells and GFP-LC3 dots per cell are shown in the left and right parts, respectively (B). Symbol * stands for p < 0.05. (C and D) U₂OS cells were transiently transfected with the indicated concentrations of Flag-PML-RARα (Flag-P-R) expression vector or the empty Flag vector (1.0 µg of each plasmid was transfected in C) for 24 h and then treated with or without pepstatin A (10 µg/ml) and E64d (10 µg/ml) (C), 3-MA (10 mM) (D) or an equal volume of the vehicle for an additional 4 h. Cell lysates were harvested and analyzed by protein gel blot with specific antibodies. Relative LC3-II expression in (C and D) was determined by the ratio of the densitometric value of LC3-II relative to the empty-transfected controls with vehicle treatment. All experiments were repeated three times with similar results, and values are shown as means with bar as SD of three independent experiments.