Autophagy is activated by ATRA and ATO in NB4 cells. (A) NB4 cells were treated with or without ATRA (1μM) for 1, 2, or 3 days (D1, D2, and D3, respectively), followed by IB analysis by the use of anti-LC3 and antitubulin antibodies. (B) Cell lysates from NB4 cells treated with or without ATO (1μM) for the indicated time were subjected to immunoblotting by the use of anti-LC3 and anti-tubulin antibodies. (C) NB4 cells with or without ATRA (1μM) or ATO (1μM) for 24 hours were stained with anti-LC3 (red) and anti-p62 (green) antibodies. Cells were examined by confocal microscopy. Scale bars, 10 μm. (D) The number and size (0.02-1.00 μm2) of p62- and LC3-positive structures were estimated by the use of the ImageJ quantification program from 2 separate experiments (total 150 cells). The graphs show average number of particles per cell ± SEM. (E-F) NB4 cells treated or not with ATRA or ATO were fluorescently labeled with antibodies against (E) PML (red) and LC3 (green) or (F) PML (red) and p62 (green). Cells were examined by confocal microscopy. Phase contrast images in grayscale are shown to visualize the nuclear boundary. Scale bars, 10 μm.
