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. 2021 May 8;12(5):464. doi: 10.1038/s41419-021-03730-8

Fig. 2. CXCR4 inversely regulates the expression and function of DR5.

Fig. 2

CXCR4 overexpressing or control stable MCF7 cells were harvested for protein extraction and analyzed for the expression of apoptotic genes by utilizing proteome profiler apoptosis array of individual western blot analysis. A Chemiluminescent image of the expression of 35 apoptosis-related genes with positive and negative controls in duplicates for control and CXCR4 overexpressed cells was shown (left panel). The enlarged images of selected apoptotic protein (DR5) found to be markedly altered in the proteome profiler array (middle-upper) and spot coordinates (middle-lower) were shown. B Heatmaps depicting differentially regulated proteins in CXCR4 overexpressing and control stable MCF7 cells. C Immunoblot analysis of DR5 protein in CXCR4 overexpressing or control MCF7 cells; β-Actin was used as an internal protein loading control. D CXCR4 overexpression and control MCF7 cells were either stained with PE-conjugated anti-human DR5 or PE tagged isotype (IgG) control antibodies, and cell surface expression of DR5 was analyzed by histogram overlays using FACS. E Immunoblot analysis of DR5 protein in CXCR4 knockdown or control HT-29 cells; β-Actin was used as an internal protein loading control. F CXCR4 knockdown and control HT-29 stable cells were stained either with PE-conjugated anti-human DR5 or PE tagged isotype control antibodies. Cell surface expression of CXCR4 was analyzed by histogram overlays using FACS. G Chili tagged CXCR4 knockdown and control HT-29 stable cells were mixed equally and subjected to FACS analysis at Day 0 and 3 days after recombinant human TRAIL (50 ng/mL) treatment. H CXCR4 knockdown and control stable cells were treated with different concentrations of TRAIL (6.25, 12.5, 25, 50, 100 ng/mL) for 48 h, and cytotoxicity was measured by SRB assay. Percent cell viability was tabulated. Columns, an average of triplicate readings of samples; error bars ± S.D. **p < 0.01; #, p < 0.05, compared to TRAIL-treated control cells. I Control and CXCR4 knockdown HT29 cells were treated with Rh-TRAIL for 24 h and stained with FITC conjugated Annexin-V. Histogram overlays show the Annexin-V positive cells. J Immunoblot analysis of cleaved PARP and Caspase-8 in 24 h post vehicle or TRAIL-treated (10, 20, and 40 ng/mL) control and CXCR4 knockdown HT-29 cell lysates; 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.