Figure 4.

Autophagy is elevated in the MTA1-overexpressing MCF7 cells. (A) The MCF7 cells that stably expressed MTA1 were pretreated with 20 nM bafilomycin A₁ (Baf) for 0.5 h and then treated with 5 μM 4OHT for an additional one h prior to examination of autophagy flux. The expression of LC3 was determined by western blotting (left). The LC3-II level was quantified from band intensity using ImageJ and expressed as relative to the level in the control cells without treatment. Values were normalized by the intensities of the corresponding TUBA band. Data are presented as the mean±SEM (n = 3). *, P< 0.05 and ***, P< 0.001 (right). (B) The MCF7 stable cells were infused with Ad-mCherry-GFP-LC3 for 24 h and then treated with 5 μM 4OHT for another 24 h, or treated with 100 nM Baf another 2 h. Then, cells were visualized with a confocal microscope. The autophagosomes (yellow in merge) and autolysosomes (red in merge) in each cell were counted (n = 50 cells/sample). Data obtained from 1 of 3 independent experiments with similar results were presented. Data are presented as the mean±SEM. ***, P< 0.001 (right). (C) Transmission electron microscopy images of the MTA1-overexpressing MCF7 cells treated with 5 μM 4OHT for 24 h. Red arrowheads indicate autophagic vacuoles. (D) The MTA1-overexpressing MCF7 cells and the control cells were transfected with siATG7 or treated with 1 μM HCQ. Then cells were treated with 1 μM 4OHT for the indicated time periods and the number of viable cells was counted using a hemocytometer. Cell numbers were presented as the mean±SEM from duplicate plates and data obtained from one of 3 independent experiments with similar results are presented. ***, P< 0.001.