Figure 7. MMV652103 induces autophagy in MCF7 breast cancer cells. (A) Representative phase-contrast photomicrographs of MCF7 cells treated with vehicle (DMSO) and IC₅₀ MMV652103 for 48 h. Yellow arrows indicate large vacuolar structures reminiscent of autophagic vesicles. (B) Representative confocal images (100X; EVOS XL AMEX1000 Core Imaging System; red scale bar = 100 µm) of MCF7 cells treated for 48 h with MMV652103 and stained with acridine orange (AO) which depicts the presence of acid vesicular organelles (AVOs, yellow to orange puncta). (C) Representative maximum intensity projection confocal immunofluorescence images (630X; Zeiss LSM 510; scale bar is 10 μm) from three independent experiments of MCF7 treated with IC₅₀ and 2 x IC₅₀ MMV652103 or vehicle for 48 h and incubated with LC3 primary antibody, fluorophore conjugated Cy3 secondary antibody and nuclei were stained with DAPI. (D) Western blotting of proteins from MCF7 cells treated with MMV652103 at IC₅₀ and 2 x IC₅₀ concentrations with LC3ß cleavage (LC3-I and LC3-II) analyzed at the indicated timepoints. (E) Cells transfected with non-silencing siRNA (sictrl) or LC3 specific siRNA (siLC3) and treated with MMV652103 at the IC₅₀ concentration for 24 h and western blot analysis performed for the indicated proteins. For all western blot analysis total p38 was used as a loading control and densitometry readings were obtained using ImageJ. Protein expression levels are represented as a ratio of protein of interest/loading control normalized to a control sample. Blots are representative of at least three independent experiments.