Salinomycin Induces Autophagy in Colon and Breast Cancer Cells with Concomitant Generation of Reactive Oxygen Species
Figure 4
Induction of autophagy by salinomycin.
(A) Electron microscopic images of MCF-7 cells 48 hours after treatment with 10 µM salinomycin or solvent control. Note the vacuoles formed in the cytoplasm (arrow). (B) Salinomycin induced uptake of monodansylcadaverine (MDC) in SW620, RKO and MCF-7 cells 8 hours after salinomycin treatment, in comparison to doxorubicin. Cells with a punctate staining pattern were counted. (*: p<0.05; **: p<0.01, ***: p<0.001 by t-test, compared to the solvent control). (C) Confocal microscopy of cells transfected with GFP-LC3 16 hours after treatment as indicated (sali: 2.5 µM salinomycin). Upper row: MCF-7, bottom row: SW620. (D) Induction of the autophagy-related genes Beclin-1, ATG7, and ATG12, and processing of LC3B after salinomycin treatment of MCF-7. Cells were treated for the indicated durations with 2.5 µM or 10 µM salinomycin, and analyzed for autophagy-specific markers by Western blot. β-actin was used as a loading control. (E) Expression of the autophagy-related gene Beclin-1, and processing of LC3B after salinomycin treatment in SW620. Cells were treated for the indicated durations with 2.5 µM salinomycin, and analyzed for autophagy-specific markers by Western blot. β-Actin was used as a loading control. (F) Processing of LC3 in SW480 after salinomycin treatment in the presence or absence of 0.25 mg/ml catalase and 20 µM SP600125. Cells were treated with 2.5 µM salinomycin for the indicated duration; the relative level of LC3-II in comparison to tubulin is indicated below the figure.