Fig. 7

BRD4 inhibitory natural compound 3',4',7,8-tetrahydroxyflavone (THF) regulated mammosphere formation derived from MDA-MB-231 and ARV-825 inhibits mammosphere formation derived from HCC1937 through the inhibition of BRD4/PD-L1/RelB. A Structure of THF. B Inhibitory effect of THF on the proliferation of MDA-MB-231 cells. The cells were cultured with the indicated concentration range of THF for 24 h. Cell viability was measured using the MTS assay. C Inhibitory effect of THF on mammosphere. The mammospheres derived from MDA-MB-231 cells were treated with the indicated concentrations of THF for 7 days. Mammosphere images (right) were taken at ×10 magnification. D Protein expression regulation by THF in mammospheres. The protein expressions of c-Myc, PD-L1, and RelB were detected by Western blot. The cells were treated with 100 µM THF for 24 h. E Protein expression regulation of THF in nuclear and cytosolic fractions of mammospheres. The protein expressions of c-Myc, PD-L1, and RelB were detected by Western blot. The cells were treated with 100 µM THF for 24 h. F Transcriptional regulation of THF on mammosphere. The cells were treated with 100 µM THF for 18 h. mRNA levels of PD-L1, RelB, and IL-6 were analyzed using reverse-transcription quantitative polymerase chain reaction. G Cytokine profiling in mammospheres. Cytokine profiling was performed at THF (100 µM)-treated mammosphere culture medium. The amount of IL-6 was quantified using a flow cytometer. H Proliferation assay using ARV-825 on breast cancer cell line, HCC1937 cells. The cells were cultured with an increasing concentration range of ARV-825 for 24 h. I Effect of BRD4 degrader ARV-825 on mammosphere formation. ARV-825-treated cells reduced mammosphere formation, as shown in the photos and graphs. Experiment values are represented as the mean ± SD of triplicates. J CSCs marker, ALDH expression on ARV-825-treated cells. The cells were treated with ARV-825 for 24 h. ALDH expression was measured using the ALDEFLUOR assay kit and a flow cytometer. K Protein expression regulation of ARV-825 in mammosphere derived from HCC1937. The protein expressions of c-Myc, PD-L1, and RelB were detected by Western blot. The cells were treated with 0.1 µM ARV-825 for 24 h. L IL-6 level of mammospheres under ARV-825 treatment. The level of IL-6 was examined in the ARV-825 (0.1 µM)-treated mammosphere culture medium. The amount of IL-6 was quantified using 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