Figure 5. Exogenous BMP2 controls HIF-1α protein level and HIF-1α dependent transcriptional activity even under hypoxia.

(A) Representative western blot analyses of HIF-1α and REDD1; GBM-derived cells, initially expanded in 2% oxygen were treated as described in Fig. 4. (B,C) Bar graphs showing mean intensity of HIF-1α and REDD1 normalized to control at 2% oxygen (corresponding to the 0 base line)±S.E.M. comparing 6 different tumors, n = 3 for each tumor. (D) HRE-luciferase assay: GBM cells and normal SVZ-derived cells were transfected either with a HRE-firefly luciferase reporter construct or with a mutated HRE version of the same construct to evaluate aspecific effects. Along with these vectors, also a Renilla luciferase vector has been transfected in order to normalize luciferase detection. Normalization of the data to the mutated HRE vector was done and then values were calibrated to untreated cells (Control). 3 different GBM have been analyzed, n = 2 for each tumor. (E) Representative western blot analysis of HIF-1α in GBM-derived cells, initially expanded in 2% oxygen, then acutely exposed to 100 µM rapamycin alone or in combination with 10 ng/ml BMP2 for 72 hrs. β-actin was used as a loading control. 2 different GBM have been analyzed, n = 3 for each tumor.