Salinomycin Promotes Anoikis and Decreases the CD44+/CD24- Stem-Like Population via Inhibition of STAT3 Activation in MDA-MB-231 Cells
Fig 2
Salinomycin induces anoikis-sensitivity.
(A) After exposure to salinomycin (0.5–2 μM) or DMSO for 24–48 h, the sub-G1 population was examined by flow cytometry. The percentage of cells in the sub-G1 fraction (red arrow) is depicted in the plot. (B-D) MDA-MB-231 and (E-F) 4T1 cells were grown in plates that were either uncoated or coated with poly-HEMA (2-hydroxyethyl methacrylate, 10 mg/ml) for 24 h and then treated with salinomycin (0.5–2 μM) or DMSO. (B) Effect of salinomycin on the sub-G1 fraction in anchorage-dependent and -independent conditions. The graph represents the percentage of cells in sub-G1 (++ p<0.01 and # p<0.05). (C) Effect of salinomycin on levels of apoptosis-related factors in MDA-MB-231 anchorage-dependent and -independent cells. Actin was used as a loading control. Quantitative graphs of cleaved caspase-3, cleaved caspase-8, and survivin signal intensity are shown (right panel, + p<0.05 and ## p<0.01). (D) Salinomycin (2 μM, 48 h) significantly increased the number of early and late apoptotic cells in anchorage-independent growth. The graph represents the percentage of annexin V-positive cells (bottom panel, + p<0.05). (E) Effect of salinomycin on levels of apoptosis-related factors in 4T1 anchorage-dependent and -independent cells. Quantification of cleaved caspase-3 and survivin are shown (right panel, +++ p<0.001 and ### p<0.001). (F) Early and late apoptotic cells were increased following salinomycin treatment (0.5 μM, 48 h) in 4T1 anchorage-independent growth. The percentage of annexin V-positive cells are shown in the graph (bottom panel, + p<0.05). The results are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (+ p<0.05, ++ 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; NS, not significant). All experiments were independently performed at least three times (n = 3).