Fig. 6. LYN-1604 induces cell death in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 0.5, 1.0 and 2.0 μM LYN-1604, respectively, then stained with MDC and observed by fluorescence microscope. Scale bar: 50 μm (left panel), 20 μm (right panel). (B) MDA-MB-231 cells were treated with 0.5, 1.0 and 2.0 μM LYN-1604, respectively. Then, cells were collected and analyzed by flow cytometry, the MDC positive ratio was quantified. (C) MDA-MB-231 cells were treated with different concentrations of LYN-1604 for 24 h and then the expressions of Beclin-1, p62 and LC3 were detected by western blot analysis. (D) MDA-MB-231 cells were transfected with GFP/mRFP-LC3 plasmid, after co-incubation with 2.0 μM LYN-1604 in the presence or absence of 10 nM Bafilomycin A1 (BafA1), the GFP-LC3 puncta were observed by fluorescence microscope. Scale bar = 20 μm. (E) MDA-MB-231 cells were co-incubated with 2.0 μM LYN-1604 in the presence or absence of 10 nM Bafilomycin A1 (BafA1), then the expression levels of p62 and LC3 were detected. β-Actin was measured as the loading control. (F) MDA-MB-231 cells were treated with 2.0 μM LYN-1604, 3-MA (1 mM) was added 1 h before treatment of LYN-1604. After the above treatment, cell viabilities were detected by MTT assay; ***, p < 0.001 vs. LYN-1604-treated group. (G) MDA-MB-231 cells were treated with 2 μM LYN-1604 for the indicated times, apoptosis ratios were determined by flow cytometry analysis of Annexin-V/PI double staining. 3-MA (1 mM) was added 1 h before treatment of LYN-1604.
