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. 2014 Dec 12;290(5):3033–3044. doi: 10.1074/jbc.M114.615179

FIGURE 4.

FIGURE 4.

BI 2536 inhibits breast cancer stem cell (CSC) phenotype, and BMI1 overexpression overcomes inhibition of breast CSC phenotype by BI 2536. A, MCF7-B0 and MCF7-BMI1 (top panel), and MDA-MB-231-B0 and MDA-MB-231-BMI1 (bottom panel) cells were plated in serum-free mammosphere culture medium supplemented with MammoCult Proliferation Supplements (StemCell Technologies, Vancouver, Canada). After 7 days of cell plating, number of mammospheres (>50 μm) were counted, photographed under phase contrast (10× mag) and plotted. Error bars represent ± S.D. *, p < 0.05 (significant); NS, not significant. B, MDA-MB-231-B0 and MDA-MB-231-BMI1 cells were mock- or BI 2536-treated and studied for ALDH1 activity using an Aldefluor assay kit (StemCell Technologies). DEAB (ALDH inhibitor)-treated cells were used to set the background. BI 2536 treatment led to 62.45% decrease in the fraction of ALDH-positive cells (from 2.53% to 0.95%) in control MDA-MB-231-B0 cells. BMI1 overexpression partially overcomes the inhibitory effect of BI 2536 on ALDH activity as MDA-MB-231-BMI1 cells showed only 32.8% decrease in the fraction of ALDH-positive cells (from 4.42% to 2.97%). C, MDA-MB-231-B0 and MDA-MB-231-BMI1 cells were mock- or BI 2536-treated and analyzed for the expression of CD44 marker using FACS analysis.