Figure 7

mRNA and protein expressions of pro-inflammatory proteins in hypoxic cancer stem cells from GBM. (A) GBM stem cells were incubated under hypoxic conditions for 6 and 24 h. After the times indicated, the cells were processed and mRNA obtained as described in Methods. mRNA expression levels for HIF-1α, NF-kB, RAGE, P2X7R, COX2, NOS2, PTX3, CXCR4 and VEGF were determined using real time PCR. The bar graphs show fold increases in expression of the studied molecules in cancer cells with respect to control normoxic cells, set at 0. Data are presented as mean ± SD for three separate experiments, each repeated in triplicate. *, P < 0.05.(B) GBM-M and -Q stem cells were incubated under normoxia or hypoxia for the times indicated. HIF-1α content was measured in nuclear fractions by Trans-AM ELISA assay as described in Methods. *, P < 0.05.(C) GBM-M stem and differentiated cells were incubated under hypoxic conditions for 6 or 24 h. After the times indicated, cells were processed as described in Methods. Contents of HIF-1α, NF-kB, RAGE, P2X7R, COX2, PTX3 and CXCR4 were determined by western blotting. β-Actin was used as a loading control. Blots are representative of at least three separate experiments. C: control normoxic cells.