Figure 6

Inhibition of FGF, VEGF, and TGF receptors suppress NSPC-induced changes in ECs. A and B: miRNA profiling (using Affymetrix microarray analysis) of the ECs co-cultured with NSPCs for 24 hours, in the presence of 1 μM FGF/VEGF receptor tyrosine kinase inhibitor (A) and 10 μM TGF-β kinase inhibitor (B). In control samples, the miRNAs were isolated from ECs grown on the transwell inserts as monocultures (in the absence of NSPCs), and treated with the same volume of the vehicle (DMSO). Significantly (~ 2 fold) upregulated miRNAs in the inhibitor-treated ECs from EC/NSPC co-cultures, as compared to control samples are shown in red, downregulated miRNAs are depicted in blue. C: Cell lysates were prepared from: ECs grown on cell culture filters alone, 2 μl/mL DMSO was added to the medium (clear bar on the graph and lane 1 on the representative immunoblot); ECs grown in co-culture with NSPCs, 2 μl/mL DMSO was added to the medium (black bar on the graph and lane 2 on the immunoblot); ECs grown in the presence of NSPCs, medium containing 1 μM FGF/VEGF receptor tyrosine kinase inhibitor PD 173074 (grey bar on the graph and lane 3 on the immunoblot); ECs grown in the presence of NSPCs; medium containing 10 μM TGF-β kinase inhibitor (striped bar on the graph and lane 4 on the immunoblot). Immunoblots were probed with anti-phospho-mTOR antibody. Signal intensities were quantified with AlphaEaseFC densitometry software. Relative density was calculated by the ratio of the protein of interest vs actin band intensities, and displayed graphically. Values represent the mean ± S.E.M. (n = 3, p < 0.05, Student's t-test).