Salinomycin Promotes Anoikis and Decreases the CD44+/CD24- Stem-Like Population via Inhibition of STAT3 Activation in MDA-MB-231 Cells
Fig 4
Salinomycin inhibits the expression of STAT3 downstream target molecules and impairs cell migration and invasion.
(A) Effect of salinomycin (2 μM, 48 h) on cyclin D1 mRNA and protein content. Quantitative graphs of cyclin D1 mRNA and protein levels are shown (bottom panel, Student’s t-test, ** p<0.01). (B) Effect of salinomycin on subcellular localization of cyclin D1. Nuclear accumulation of cyclin D1 (1:100, green) was reduced following salinomycin (2 μM, 48 h) treatment. White arrow indicates nuclear cyclin D1; the nuclei were stained with DAPI. (C) Effect of salinomycin on cyclin D1 and survivin protein content in the presence or absence of IL-6 (0–10 ng/ml) in MDA-MB-231 cells. Quantitative graphs of cyclin D1 and survivin protein levels are shown (bottom panel, * p<0.05 and ### p<0.001). (D) MMP-2 and MMP-9 mRNA abundance was analyzed by RT-PCR analysis of total RNA isolated from DMSO- or salinomycin-treated cells (2 μM, 48 h). Quantitative graphs of MMP-2 and MMP-9 mRNA levels are shown (right panel, Student’s t-test, * p<0.05). (E) Effect of salinomycin on cell migration. After salinomycin treatment (0–10 μM), kinetic analysis of cell migration was conducted using an IncuCyte™ Live-Cell Imaging System for the indicated time durations. (a) The kinetic graph of cell migration represents the relative wound density (** p<0.01). (b) Representative images show wound closure by cell migration at 0 and 24 h in the presence or absence of 2 μM salinomycin. The black lines indicate the initial scratch areas (width, 700–800 μm) and the gray regions represent the empty space not covered by cells. (F) Effect of salinomycin on cell invasion. Images of invading cells were captured with an inverted microscope at ×200 magnification. Enlarged images from selected areas are shown. The graph represents the percentage of invaded cells (right panel, Student’s t-test, ** p<0.01). (G-H) Effect of salinomycin (0.5–2 μM, 24 h) on STAT3 and phospho-STAT3 protein levels in (G) MDA-MB-231 and (H) 4T1 cells in anchorage-dependent and -independent growth. Quantitative graph of intensity ratio of STAT3 and phospho-STAT3 is shown (bottom panel). The results are presented as mean ± SEM and were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (* p<0.05, ** p<0.01, ***p<0.001, versus anchorage-dependent DMSO control; ++ p<0.01 and +++ p<0.001, versus anchorage-independent DMSO control; # p<0.05, ## p<0.01 and ### p<0.001, versus each concentration). All experiments were independently performed at least three times (n = 3).