Measurement of autophagy and early apoptosis (Annexin-V) in anti-microRNA 21 (miR-21)-treated U373 cells.

<p>(A) A representative illustration showing the flow cytometry measurement of acidic vesicular organelles (AVOs) by acridine orange staining, representing autophagic activity 48 hours after exposure to 8 Gy of radiation. (B) Statistical analysis of independent triplicate values of the percentage of cells harboring AVO resulted in significant differences between the negative-control and anti-miR-21-treated cells (*P<0.05). (C) Western blot of the LC-3 protein, in which the appearance of the lower 1b band represents autophagosome-lysosome fusion. (D) Green fluorescent protein-labeled LC-3 protein (GFP-LC3) was stably over-expressed in U373 cells using a lentiviral vector, and GFP-LC3 granule-positive cells after irradiation (white arrow) were counted using a fluorescence microscope (10×20 high power fields; HPF). (E) The number of GFP-LC3 granule positive cells was counted as a percentage of total observed cells. Statistical analysis of the average percentage of ten HPFs showed significant differences (*P<0.05). (F) The flow cytometry measurements of Annexin-V staining representing apoptotic indices. The increased early apoptotic index 48 hours after 8 Gy of irradiation in anti-miR-21-treated U373 cells compared to that in negative control-treated cells (**p<0.01). Augmented apoptosis by transfection of anti-mir21 was neutralized by a 48 hour incubation with 10 mM 3-MA, an autophagy inhibitor. Each bar on a column represents the standard error of the mean for triplicate experiments.