Fig. 1

BRD4 inhibitor, (+)-JQ1, reduces triple-negative breast cancer growth and mammosphere formation. A Effect of JQ1 on the proliferation of MDA-MB-231 cells. The cells were cultured with the indicated concentration range of JQ1 for 24 h. Cell viability was measured using the MTS assay. B Inhibitory effect of mammosphere formation by JQ1. Treatment with 0.5 µM JQI reduced mammosphere formation to 20%. C Breast CSC marker, CD44⁺/CD24⁻ expression in MDA-MB-231 cells. The cells were treated with 1 µM JQ1 for 1 day. CD44⁺/CD24⁻ expression was evaluated using a flow cytometer. D CSC marker, ALDH expression in MDA-MB-231 cells. The cells were treated with 1 µM JQ1 for 1 day. ALDH expression was measured using the ALDEFLUOR assay kit and a flow cytometer, as described in the “Materials and methods” section. E CSC marker gene expressions in MDA-MB-231 cells treated with JQ1. The cells were treated with 0.5 µM JQ1 for 18 h. The mRNA levels of CD44, c-Myc, OCT4, and SOX2 were measured by reverse-transcription quantitative polymerase chain reaction. F Inhibitory effect of JQ-1 on mammosphere growth. JQ1-treated mammospheres were divided into single cells, and equal numbers of cells were cultured. The number of cells was analyzed daily for 3 days by counting. G Difference in BRD4 protein expression in breast cancer cells and mammospheres. BRD4 protein expression was analyzed in cancer cells and mammospheres derived from MDA-MB-231 and MCF-7 cells by Western blot, as described in the “Materials and methods” section. H Effect of BRD4 on mammosphere formation. After BRD4 knockdown using siRNA, mammosphere formation was reduced, as shown in the photos and graphs. The knockdown of BRD4 was verified by Western blot, and images of the mammosphere (right) were taken at ×10 magnification. I CSC marker, CD44⁺/CD24⁻ expression on BRD4-knockdown cells. The cells were treated with siBRD4 for 2 days. CD44⁺/CD24 expression was evaluated using a flow cytometer. J CSC marker, ALDH, expression on BRD4-knockdown cells. The cells were treated with siBRD4 for 2 days. ALDH expression was measured using the ALDEFLUOR assay kit and a flow cytometer. Experiment values are represented as the mean ± SD of triplicates. Compared with the control as determined by student’s t-test or one-way ANOVA with Dunnett’s multiple comparisons tests, * p < 0.05