The inhibitor 3-MA suppressed autophagy development in cells with ectopic Bβ2. (A) Autophagosome formation by acridine orange staining. Cells plated on coverslips in 6-well plates were either pretreated with (+) or without (-) autophagy inhibitor 3-MA before being given 50 nM tBHP for 48 h. The cells were then fixed and stained with acridine orange (green) and analyzed by fluorescence microscopy. The formation of autophagosome as shown in puncta fluorescence (red) can be suppressed by 3-MA. The presence of autophagy was found mainly in 100% of the cells after treatment, in which more than 100 cells were observed under each condition. Scales, 50 μm. Cells as pointed out by arrows were amplified as shown in the inset (2×). (B) Western blot probed with LC3 antibody The development of autophagy by tBHP in the presence (+) or absence (-) of 3-MA was analyzed by western blot analysis by incubating the blot with LC3 antibody. Levels of the 18-kDa LC3-I is visualized in both Bβ2 and Bβ1 clones. Elevated LC3-II, the 16-kDa form of LC3 specific for membranes of autophagosomes by tBHP in Bβ2 clones was suppressed by 3-MA.