Figure 3. Thalidezine induces autophagic flux in HeLa cells.

(A) Thalidezine induces LC3-II conversion in the presence of lysosomal inhibitors. HeLa cells were treated with 10 μM of thalidezine in the presence or absence of 10 μg/mL lysosomal protease inhibitors (E64d and pepstatin A) for 24 h. Immunoblot for LC3-I, LC3-II, and β-actin detection (left). LC3 conversion was expressed as fold change relative to the DMSO-treated negative control (right). Uncropped blots images are shown in Supplementary Figure 4C. (B) tfLC3 fluorescence detection pattern of thalidezine. HeLa cells were transfected with mRFP-GFP-LC3 plasmids and treated with 10 μM of thalidezine (Tha) for 0-24 h. Representative micrographs of cells with GFP-LC3 puncta (green channel), mRFP puncta (red channel), and merge images are shown (upper-left and right panel). Scale bar = 15 μm, 60X. Each correlation plot was derived from the field shown in the fluorescence image (lower-right panel). Histogram represented the quantification of the percentage of colocalization between mRFP and GFP signal (lower-left panel). **, P ≤ 0.01; ***, P ≤ 0.001. Data were mean value ± S.D of at least three independent experiments.