Fig. 7
Effect of rapamycin, chloroquine, an ATG7-specific siRNA or an scrambled control siRNA on long lived protein turnover (flux assay), LC3-II levels, or lysosome and doxorubicin localization in MCF-7CC10 and MCF-7DOX2–10 cells. The flux assay was conducted to examine the overall hydrolysis of long lived proteins through autophagy after cells are treated with the autophagy activator rapamycin (Rap), the autophagy inhibitor chloroquine (CQ) (A, upper panel), or siRNAs specific for the ATG7 gene or a scrambled control (B, upper panel). Immunoblot analysis was used to assess LC3-II protein accumulation in the cells that were treated with either rapamycin (Rap) or chloroquine (CQ) for 24 h compared to a control solution DMSO (vehicle; veh) (a, lower panel). The efficiency of Atg7 protein knockdown by siRNA interference (b, lower panel) compared to scramble control (scr) was also assessed in this experiment using immunoblot analysis with anti-Atg7 antibodies. Confocal microscopy examination (panel c) was also performed to show the effect of chloroquine on the subcellular distribution of lysosomes (green) and doxorubicin (red) in MCF-7CC10 cells (c, left) and MCF-7DOX2–10 cells (c, right). MCF-7 cc10 cells were treated with 10 μM of chloroquine and 2 μM of doxorubicin for 8 h, and MCF-7DOX2-10 cells were treated with 10 μM of chloroquine and 2 μM of doxorubicin for 48 h. The images in panel C represent one of the 10 microscopic photos from two sets of separately stained slides in two independent experiments. The staining and phenotype were very consistent throughout 100 viewed cells