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. Author manuscript; available in PMC: 2019 Feb 15.
Published in final edited form as: Clin Cancer Res. 2017 Nov 16;24(4):821–833. doi: 10.1158/1078-0432.CCR-17-1628

Figure 3. mAb428.2 inhibits fibulin-3 signaling in glioblastoma cells.

Figure 3

A) U251MG stably expressing a Notch-dependent reporter (CBF1:Luc) were transfected with fibulin-3 or control cDNAs and after 24h treated with 50 μg/ml of mAb428.2. The antibody inhibited the enhancing effect of fibulin-3 on Notch activity within 6h. The inset shows the level of fibulin-3 overexpression in the cells, quantified as shown in Figure 1C. B) Same experimental design as in (A) but mAb428.2 was added at different concentrations for 6h. Fibulin-3-enhanced Notch activity was completely blocked by mAb428.2 at concentrations above 30 μg/ml. C) Same experimental design as in (A), using cells stably expressing a NF-κB-dependent reporter (NKRE:Luc). mAb428.2 inhibited the enhancing effect of fibulin-3 on NF-κB activity within 6h. Firefly Luciferase activity of the reporters was normalized to a transfected Renilla Luciferase control as indicated in the Methods. Significant differences in (A) and (C): *** p<0.001 (2-way ANOVA). D) U251MG cells were transfected with fibulin-3 or control cDNAs and lysed after 24h; the lysates were incubated with an ADAM17 fluorogenic substrate to measure enzymatic activity in presence or absence of mAb428.2. Fibulin-3 overexpression increased ADAM17 activity but this effect was completely abolished when mAb428.2 (50 μg/ml) was present in the reaction mixture. Treatment of cells or lysates with non-immune IgG in experiments (A–D) had no effects on reporters or enzymatic activity. E) U251MG cells transfected with NICD cDNA (for 24h) to activate the Notch reporter, or treated with TNFα (10 ng/ml, 6h) to activate the NF-κB reporter, showed no changes in reporter activity when they were co-transfected with fibulin-3 cDNA or incubated with antibodies (50 μg/ml). F) U251MG cells and two GSC cultures (GBM09 and GBM34) were treated with mAb428.2 (50 μg/ml) or a control IgG for 24h and then processed to measure mRNA expression of Notch- and NF-κB-dependent genes. mAb428.2 reduced the expression of the four tested genes compared to control IgG treatment (* p<0.05; + p<0.01; # p< 0.001, by 2-way ANOVA for each cell type).