Fig. 6
From: BRD4/nuclear PD-L1/RelB circuit is involved in the stemness of breast cancer cells
RelB/p65 complex regulated IL-6 transcriptional expression. A Effect of calcitriol and caffeic acid phenethyl ester on interleukin (IL)-6 and IL-8 of mammospheres. Mammospheres were treated with 40 µM calcitriol or 20 µM caffeic acid phenethyl ester for 18 h. mRNA levels of IL-6 and IL-8 were analyzed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). B Effect of BRD4 inhibitor on IL-6 gene expression on mammosphere. Mammospheres were treated with 1 µM JQ1 or 0.5 µM ARV-825 for 18 h. mRNA levels of IL-6 were analyzed by RT-qPCR. C Effect of PD-L1 inhibitor on IL-6 gene expression on mammospheres. Mammospheres were treated with 4 µM verteporfin for 18 h. mRNA levels of IL-6 were analyzed by RT-qPCR. D ChIP assay on the promoter of IL-6. The binding site of NF-kB on IL-6 promoter is shown in Fig. 6D. Mammospheres were treated with 40 µM calcitriol. ChIP analysis used an antibody against p65 or RelB. The negative control used was IgG. E Cytokine profiling in mammospheres. Cytokine profiling was performed at the drug-treated mammosphere culture medium, and the drug concentrations were as follows: 1 µM JQ1, 0.5 µM ARV-825, 4 µM verteporfin, 40 µM calcitriol, or 20 µM caffeic acid phenethyl ester. The amounts of IL-6 and IL-8 were quantified using a flow cytometer. F Rescue experiment using PD-L1 overexpression plasmid to confirm the existence of the BRD4/PD-L1/RelB/IL-6 axis. Protein expression regulation by ARV-825 in breast cancer. The PD-L1 expression plasmid vector, pEGFP-N1/PD-L1 was transfected into MDA-MB-231 cells and the transfected cancer cells were treated with 0.5 µM ARV-825 for 24 h. The protein expressions of BRD4, PD-L1, and RelB were detected by Western blot. Cytokine profiling was performed at ARV-825-treated cancer cell culture medium. The amount of IL-6 was quantified using a flow cytometer. Experiment values are represented as the mean ± SD of triplicates. 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