Figure 3.

The effects of PLZF-RARα and NPM-RARα fusion proteins on autophagy. U₂OS cells were co-transfected with GFP-LC3 and hcRed-PLZF-RARα, DsRed-PML-RARα, or the corresponding empty vectors (HcRed or DsRed), or with Myc-LC3 and CFP-NPM-RARα or the CFP vector. After transfection for 24 h, the cells were stained with anti-Myc antibody or directly analyzed by confocal microscopy. The Myc-LC3 signal was imaged on the red fluorescent protein (RFP) channel, and the CFP signal was obtained on the CFP channel. (A) Representative images of the cells transfected with the indicated constructs were shown. Arrowheads indicate cells with the expression of proteins as labeled. (B) The percentage of GFP-LC3 puncta-positive cells (left part) and the total number of GFP-LC3 dots per cell (right part) were calculated. The symbol *indicates a p value of less than 0.001 compared with the cells co-transfected with DsRed and GFP-LC3 plasmids. (C) After a transient transfection with the indicated plasmids, U₂OS cells were extracted and detected by protein gel blot. The transfected expressions of APL-related fusion proteins were confirmed by a RARα antibody. (D) U937/PLZF-RARα cells were treated with 100 µM ZnSO₄ for the indicated hours and the cell lysates were harvested for immunoblotting. Relative LC3-II in (C and D) was determined by the ratio of densitometric value of LC3-II relative to the corresponding empty-transfected or the untreated controls. All experiments were repeated three times with similar results, and all values were shown as means with bar as SD of three independent experiments.