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. 2013 Dec 27;6(2):278–294. doi: 10.1002/emmm.201303373

Figure 5.

Figure 5

A Sixteen h after plating, cells were cultured under normoxic (−) or hypoxic (+, 1% O2) conditions for 24 h (MDA-MB-231 and Hs578T) or 6 h (shCtrl-3 and shA8-20 clones). WCEs were subjected to WB for ADAM8 (LSBio antibody). Representative blots are shown ( n = 3).

B ADAM8 expression in mouse mammary tumors derived from shCtrl-3 or shA8-20 cells was analyzed by immunohistochemistry. H&E staining was performed in parallel. Representative panels are shown ( n = 7/group). Bar: 100 μm.

C Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections from shCtrl-3 and shA8-20 groups ( n = 7/group). Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (3 peritumoral hot spots/slide). P: Peritumoral area, T: Tumor. Bar: 100 μm. * P = 0.01, Student's t-test.

D Pearson's pairwise correlation plot shows a significant positive correlation between ADAM8 and CD31 (PECAM1) mRNA expression in tumors from patients with basal-like breast cancer (GenExMiner microarray database). r: correlation ratio. P < 0.0001, Student's t-test.

E, F HUVECs were subjected to tube formation assays in the presence of conditioned medium from the indicated shA8 and shCtrl clones, or obtained in absence of tumor cells (−). Values for branch points and closed networks (polygons) are given as averages of nine fields ± s.d. Branch point: * P = 1.3E-6 versus shCtrl-3, * P = 3.2E-9 versus shCtrl-5, ** P = 6.9E-29; Polygons: * P = 5.2E-6 versus shCtrl-3, * P = 1.3E-6 versus shCtrl-5, ** P = 6.2E-24; Student's t-test; n = 4 (E). Representative images from 4 independent experiments are shown. Bar: 30 μm (F).

G Conditioned media from two shCtrl and two shA8 clones were subjected to a Human Angiogenesis antibody array. Expression levels of the detected proteins were quantified using ImageJ and the angiogenesis mediators significantly downregulated by more than 2-fold in shA8 clones are presented as mean of the two clones ± s.d. Fold change (F.C.) and P-values are given, Student's t-test.

H Conditioned serum-free medium from shCtrl and shA8 clones was analyzed by WB for VEGF-A. A representative blot is shown ( n = 3).

I VEGF-A in the conditioned serum-free medium from shCtrl-3 or shA8-20 clones transfected with the indicated ADAM8 forms or empty vector (EV) DNA was assessed by WB (lower panel). The quantification of average levels from 3 experiments is presented as percent relative to the shCtrl set to 100%.