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. 2013 Jun 4;8(6):e66281. doi: 10.1371/journal.pone.0066281

Figure 4. Induction of preLOX and LOX-PP inhibits cell proliferation in A673 Ewing sarcoma cells.

Figure 4

A) A polyclonal population and a representative clone of A673/TR/empty (control cells), A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were maintained during 25 days in standard culture medium or in culture medium containing doxycycline (DOX, 1 µg/ml) to induce the expression of the corresponding proteins. Cumulative number of population doubling were determined by counting cells at different periods of time and plotted versus time. B) Representative clones of A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) for 5 days to induce the corresponding proteins and cell growth quantified by CellTiter-Fluor assay. The figure shows the mean±SD of one out of three independent experiments done in triplicate with similar results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005 versus unstimulated cells). C) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) and in the absence or in the presence of the irreversible antagonist of lysyl oxidase activity β-aminopropionitrile (β-APN, 500 µM). The figure shows the mean±SD of one out of two independent experiments done in triplicate with equivalent results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005, ns: not significant). D) A673 cells were incubated during 72 hours with conditioned media derived from A673/TR/LOX-PP cells cultured in absence of doxycycline (control) or conditioned media derived from the same cells cultured in presence of doxycycline to induce the expression of LOX-PP (LOX-PP rich media). The figure shows the mean±SD of two independent experiments done in triplicate. Data are shown as percentage versus cells incubated with control media, which were arbitrarily set to 100 (*P<0.05). LOX-PP rich media significantly reduced cell proliferation of A673 cells.