Fig. 3

BRD4 degrader ARV-825 inhibited PD-L1 expressions, and PD-L1 regulated mammosphere formation. A PD-L1 protein expression in breast cancer cells and mammospheres. PD-L1 protein expressions were 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. B Transcriptional regulation of PD-L1 genes by BRD4 inhibitor JQ1 and ARV-825-treated MDA-MB-231 cells and mammospheres. The cells were treated with 0.5 µM JQ1 and 0.5 µM ARV-825 for 18 h. The mRNA level of PD-L1 was measured using reverse-transcription quantitative polymerase chain reaction. C PD-L1 protein levels in mammospheres after treatment of the BRD4 inhibitor and ARV-825. The mammospheres were treated with 1 µM JQ1 and 0.5 µM ARV-825 for 24 h. D, E Fractional analysis of PD-L1 protein expression in mammospheres. Cell lysate was fractionated using an isolation kit, as described in the “Materials and Methods” section. The mammospheres of MDA-MB-231 cells were treated with 1 µM JQ1 or 0.5 µM ARV-825 for 24 h. The fractions were analyzed by Western blot with PD-L1 antibody, and subcellular location markers were detected with antibodies (α-tubulin, ATPase, Lamin-B, and vimentin). F PD-L1 reporter luciferase assay using MDA-MB-231 cells and mammospheres. PD-L1 reporter plasmid was transfected into cancer and mammosphere, and cells were then treated with 1 µM JQ1 and 0.5 µM ARV-825 for 24 h. The cells were lysed, and luciferase was assayed as described in the “Materials and methods” section. G Chromatin immunoprecipitation (ChIP) assay on the promoter of PD-L1 gene using anti-BRD4. The binding site of BRD4 on the CD274 (PD-L1) gene is shown in Fig. 3G. Mammospheres were treated with 1 µM JQ1 or 0.5 µM ARV825. ChIP analysis using an antibody against BRD4 and the negative control IgG. H Effect of PD-L1 on mammosphere formation. Cultured MDA-MB-231 cells were treated with siPD-L1 for 2 days. The mammospheres derived from siPD-L1 cells were cultured for 7 days. PD-L1 knockdown was verified by Western blot. 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