Figure 7
A recombinant protein comprising amino acids 1 to 79 of human PRL inhibits the proliferation of endothelial cells. (a) Western blot analysis of the conditioned media (CM) obtained from HEK293T/17 cells stably transduced with lentiviral vectors encoding the first 79 residues (~9 kDa) or the first 123 residues (~14 kDa, a control vasoinhibin) of human PRL. The conditioned media of HEK293T/17 cells transduced with the empty lentiviral vector was the negative control (Neg). (b) Western blot of the same conditioned media shown in (a) after treatment with Peptide N-glycosidase F (PNGase). Numbers on left indicate the molecular weight of marker proteins. (c) Representative fields of bovine pulmonary artery endothelial cells (CPAE) stained for DNA (Hoechst), DNA synthesis (EdU), and the overlay of both reactions. Cells were incubated for 24 h with or without basic fibroblast growth factor (bFGF) in combination or not (Ctl) with conditioned media without (Neg) or with the 79- (Vi79) or the 123- (Vi123) residue vasoinhibins. Scale bar, 100 μm. (d) Cell proliferation was quantified by the Edu-click reaction and expressed relative to the total number of cells in the field. Values are means ± SEM of 3 independent experiments (*p < 0.001). (e) Cell proliferation was evaluated by the [3H]-thymidine incorporation to DNA in CPAE incubated for 24 h with bFGF together or not (Ctl) with conditioned media lacking (Neg) or containing with the Vi79 or the Vi123 residue vasoinhibins. Values are means ± SEM of triplicate determinations (*p < 0.001). (f) Dose-dependent inhibition of bFGF-induced proliferation of bovine umbilical vein endothelial cells (BUVEC) incubated for 24 h with bFGF in combination or not (Ctl) with conditioned media without (Neg) or with the Vi79 or the Vi123. Cell proliferation was quantified by the Edu-click reaction and expressed relative to the total number of cells in the field. Values are means ± SEM of triplicate determinations.
