Glioblastoma Cell-Secreted Interleukin-8 Induces Brain Endothelial Cell Permeability via CXCR2
Figure 2
The glioblastoma cell secretome contains high levels of IL-8.
A) The status of the NF-κB (pIκBα and IκBα) and MAPK (pERK1/2, ERK2, pp38, pJNK, and JNK) signaling pathways were assessed in lysates from serum-starved hCMEC/D3 (D3) and U87 by western-blot. B) Promoter activity of both AP-1 and NF-κB in D3 and U87 were determined by luciferase reporter assays. C) Immunofluorescent staining for NF-κB p65 (green) and c-Jun (green) was performed as readout of NF-κB and AP-1 signaling activity, respectively, in serum-starved D3 and U87. DAPI (red) was used to stain nuclei, scale bars: 10 µm. D) The angiogenic secretome profile was determined in U87 conditioned medium (CM) using antibody array, IL-8 and its closely related family member, Gro-α, are indicated. E) The mRNA levels of IL-8, CXCR1 and CXCR2 cells were determined in U87 and D3 by RT-PCR. β-actin was used as a control for loading and PCR efficacy. F) IL-8 secretion in conditioned media from U87 and D3 was examined through ELISA. G) Expression of the IL-8 transcript was examined in D3, U87 and additional glioblastoma cell lines (GBM, U138, U251 and LN229) by RT-PCR. H) Similarly, IL-8 secretion was measured in these cell lines by ELISA. One out of three independent experiments is shown. T test on 3 independent experiments: *p<0.05; **p<0.01.