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Fig. 7. Modulation of cell proliferation by agonists of P2Y and P1 receptors. (A,B) Immunocytochemistry (representative images). Neurospheres from C57BL/6N mice were dissociated and expanded in the presence of either 20 ng/ml EGF and 10 ng/ml FGF2 (A) or 5 ng/ml EGF and 2.5 ng/ml FGF2 (B). Equal expression of nestin was observed in 4-day-old neurospheres whether cultured at high (A, H-nestin) or reduced (B, R-nestin) growth factor concentrations. (C-F) The daily addition to reseeded cells of adenosine (C), the P2Y₁-receptor agonist ADPßS (D), the P2Y₂ receptor agonist UTP (E) or of ADPßS and UTP (F) (50 µM each) had no effect on nestin expression. Scale bars: 10 µm. (G) Agonist-dependent effect on cell proliferation after daily application of ADPßS+UTP, ADPßS, UTP, UDP, ,ßmeATP, BzATP (50 µM each) or adenosine (ADO) (1 µM to 50 µM) to cells dissociated from neurospheres and expanded in the presence of reduced growth factor concentrations. The cell number was determined after 4 days. (H) Cell proliferation in neurospheres derived from wild-type (+/+) and P2Y₁-receptor knockout (-/-) mice. First, the cell number obtained after 4 days was compared between wild-type (control, 100%) and P2Y₁-knockout mice (-/-) (both C57BL/6J). In addition, the effect of the P2Y₁-receptor antagonist MRS2179 (100 µM) on neurospheres derived from wild-type mice was analyzed under two different experimental conditions. In one case [MRS2179(d4)], the antagonist was added immediately after reseeding and then daily for 4 days. In the other case [MRS2179(d7)], neurospheres were first cultured for 4 days in the presence of high growth factor concentrations, washed and reseeded at reduced growth factor concentrations. A single dose of MRS2179 (100 µM) was then applied and the cell number was determined at 7 days. (I) Comparison of the proliferative effect of P2-receptor agonists on neurosphere cells from wild-type (+/+) and P2Y₁-receptor knockout (-/-) mice. Processing of cells was as for G. Values in G-I represent mean±s.d., n=3-7, ^*P<0.05, ^**P<0.01. The average cell number derived from a single well in the control experiments (100%) amounted to 4.0±1.2x10⁶ (s.d.) for G, to 2.8±1.3x10⁶ (s.d.) for H, and to 3.3±1.3x10⁶ (s.d.) for I.

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