Figure 4. Upregulation of various cytokines in culture filtrates of hUCBSC challenged with 4910 GSC. Conditioned media was collected from hUCBSC and hUCBSC challenged with 4910 GSC. The culture filtrates were incubated on the array membrane and visualized by ECL reagents as described in Materials and Methods. The orange and green boxes represent the positive and negative controls, respectively. (A) Cytokine antibody array analysis of hUCBSC. (B) Cytokine antibody array analysis of challenged hUCBSC. (1:GRO; 2:GRO-α; 3:IL-6; 4:IL-8; 5:IL-10; 6:MCP-1; 7:MCP-2; 8:Rantes; 9:IGFBR2; 10:Osteopontin; 11:TIMP-1; 12:TIMP-2) (C) Levels of IL-8, SDF1, MCP-1, GRO-α, and IL-6 were detected by western blotting of culture filtrates of control hUCBSC, GSC and GSC co-cultures with hUCBSC. (D) Total protein was simultaneously isolated from mouse xenografts for western blotting to determine the expression of CXCR4 and CD9. The blots were stripped and reprobed with GAPDH antibody as a loading control. (E) Measurement of the levels of GRO-α, IL-8, SDF-1 and CXCR4 by RT-PCR in U251, 5310 and 4910 GSCs co-cultured with hUCBSC. Fold changes relative to the control are shown. (F) In vitro migration assay of hUCBSC toward recombinant IL-8. Representative photomicrographs of stained Matrigels demonstrating migration of control hUCBSC and CXCR4 functionally blocked hUCBSC toward the conditioned medium substituted with 10 ng/mL recombinant IL-8. Cell migration was evaluated after staining from randomly selected fields, enumerating migrating cells (n = 3).