Fig. 5. CXCR4 protein knockdown results in compromised tumor growth and DR5 overexpression in vivo.
In total, 2 × 106 stable control (HT-29 and DLD-1) or CXCR4 knockdown (HT-29 and DLD-1) cells in 100μl PBS were injected subcutaneously in the flanks of the right or left hind leg of 4–6 weeks old Crl: CD1-Foxn1nu mice respectively. Tumor volumes were measured after regular intervals by using a caliper. Growth curves for HT-29 (A) and DLD-1 (B) are shown for tumors generated from control and CXCR4 knockdown cells; points are indicative of the average value of tumor volume ± SE (n = 5 for HT-29, n = 6 for DLD-1); *p < 0.05 compared to control tumors. C, D Upper panels represent images of tumor-bearing mice, control (right flank), and CXCR4 knockdown (left flank). Mice were sacrificed, and the respective tumors from HT29 (C) and DLD-1 (D) were harvested and shown in photographs in lower panels. Single cells from respective control and CXCR4 knockdown HT-29 (E) and DLD-1 (F) were harvested and stained with either APC-conjugated anti-human CXCR4 (CD184) or isotype control antibodies, and contour FACS plots analyzed cell surface expression of CXCR4. Harvested tumors generated from control and CXCR4 knockdown cells HT-29 (G) and DLD-1 (H) were subjected to Western blot analysis for CXCR4 and DR5. β-actin was used as protein loading control. Western Blot densitometric quantification numbers are shown above the loading control blot of all immunoblot studies.