Fig. 2
US28 signaling in enhances growth of GBM spheroids. a Inducible expression of US28 in U251-iUS28 cells. Expression of US28 was induced with doxycycline. Bottom panel shows US28 expression in HCMV Merlin-infected U251 cells. US28 was stained in fixed/permeabilized cells using polyclonal rabbit-anti-US28 antibodies directed against the C-terminus of US28. Scale bars represent 20 μm. b Whole-cell binding of 125I-CCL5 to U251-iUS28 cells shows membrane expression of US28 upon induction with doxycycline. Specific displacement of 125I-CCL5 was performed with an excess of CX3CL1. c Constitutive US28-mediated PLC activation U251-iUS28 cells upon induction of US28 expression as subsequently determined by inositol phosphate ([3H]-IP) accumulation. d US28 expression in U251-iUS28 cells induces growth of GBM spheroids. Spheroids were generated by the hanging droplet method. A representative graph of spheroid size quantification is shown. Individual spheroid sizes are plotted as percentages with mean ± SEM (n = 6 spheroids per group). e Spheroids from U251-iUS28 cells secrete VEGF upon induction of US28 expression. The data show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test)
