1. ADAM8 is induced by hypoxia and promotes angiogenesis ASixteen 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).
ADAM8 is induced by hypoxia and promotes angiogenesis ASixteen 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). BADAM8 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. CAngiogenesis 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. DPearson'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, FHUVECs 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). GConditioned 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. HConditioned serum‐free medium from shCtrl and shA8 clones was analyzed by WB for VEGF‐A. A representative blot is shown (n = 3). IVEGF‐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%.
Mathilde Romagnoli et al. EMBO Mol Med. 2013;emmm
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