Fig. 3: CXCR4 regulates DR5 transcription by differentially modulating the recruitment of transcription factors p53 and YY1 at the promoter site of DR5.

A Western blot analysis of DR5 in control and CXCR4 overexpressing MCF-7 cells after bafilomycin or MG132 treatment for 5 h; β-actin was used as the protein loading control. B, C Total RNA was isolated from CXCR4 overexpressing (MCF-7) and knockdown (HT-29) stable cells along with their respective controls and reverse transcribed. Fold change in DR5 mRNA expression was measured by RT-qPCR as described in Materials and Methods. Data are representative of three independent experiments, resulting from duplicate readings of two different samples; Columns, average value of DR5 mRNA expression; bars ± SEM. *, p < 0.05, compared with respective controls. D Western blot analysis of p53, YY1, and Sp1 in control and CXCR4 overexpressing MCF-7 cells; GAPDH or β-actin was used as the protein loading control. Western Blot densitometric quantification numbers are shown above the loading control blot of all immunoblot studies. E Fold change in mRNA expression of p53, YY1, and Sp1 in control and CXCR4 overexpressing stable MCF-7 cells was assessed by RT-qPCR; Columns, average value of p53/yy1/sp1 mRNA expression; bars ± SEM. *, p < 0.05, compared with respective control. F Diagrammatic representation for the p53 binding on activator site as well as YY1 binding on repressor site of the DR5 gene promoter region. G, H ChIP assay for the analysis of YY1 and p53 recruitment on the DR5 gene promoter in CXCR4 overexpressing and control stable MCF7 cells followed by RT-qPCR. Fold change in p53 and YY1 recruitment on the respective activator and repressor sites of the DR5 gene promoter were assessed in control and CXCR4 overexpressing MCF-7 cells. Results are representative of at least two independent experiments; Columns, an average of duplicate readings of samples; error bars ± S.D. *p < 0.05 versus control MCF7 cells.