Fig. 4

PD-L1-regulated RelB gene-regulated mammosphere formation. A Regulation of PD-L1 expression in mammosphere by verteporfin, a PD-L1 degrader. The cells were treated with 2 µM verteporfin for 18 h. The mRNA levels of PD-L1 were measured by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). The protein expression of PD-L1 was detected by Western blot. The cells were treated with 2 and 4 µM verteporfin for 24 h. B Transcriptional regulation of RelB by PD-L1 inhibition. After treatment of 2 µM verteporfin for 18 h, the mRNA levels of RelB were measured by RT-qPCR. The cells were treated with 2 and 4 µM verteporfin for 24 h, and the protein expression of RelB was detected by Western blot. C Transcriptional and translational regulations of RelB through BRD4 inhibition. The mRNA levels of RelB were measured by RT-qPCR. The cells were treated with 1 µM JQ1 or 0.5 µM ARV-825 for 18 h. The protein expression of RelB was detected by Western blot. The cells were treated with 1 µM JQ1 or 0.5 µM ARV-825 for 24 h. D Regulation of mammosphere formation by RelB. Cultured MDA-MB-231 cells were transfected with si-RelB for 2 days. The mammospheres derived from si-RelB-transfected cells were incubated for 7 days. RelB knockdown was verified by Western blot. The mammosphere images (right) were taken at ×10 magnification. E Inhibitory effect of RelB inhibitor calcitriol on mammosphere formation. Proliferation of calcitriol-treated cells was measured using the MTS assay. The cells were treated with an increasing concentration range of calcitriol for 24 h. Mammospheres derived from MDA-MB-231 cells were treated with the indicated concentrations of calcitriol for 7 days. The RelB-inhibitory effect of calcitriol was verified by Western blot. Mammosphere images (right) were taken at ×10 magnification. 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