Figure 3. Increased ALP activity and mineralization of pulp cells after treatment with NO in mineralization-promoting (MP) medium. (A) The pulp cells cultured for 7 days in normal (Normal) and MP medium (MP) in the presence of various concentrations of NOC-18 were stained with ALP activity. (B) ALP activity was measured in cells cultured for 7 days in normal growth medium (circle) or in MP medium (square) in the presence of various concentrations of NOC-18. *Significantly higher than the value obtained in the absence of NOC-18 (n = 4). (C) ALP activity of cells was determined after culture for 7 days without cytokine or with 50 ng/mL BMP-2, 5 ng/mL TGF-ß1, or 10 ng/mL FGF-2 in MP medium in the absence or presence of 5 mM L-NAME. * and indicate that the values are significantly higher and lower, respectively, than the control without cytokine and L-NAME (the column at the far left). #Significant difference. Data are from 4 experiments. (D) The cells were stained with Alizarin red after 14-day culture in MP medium in the absence (Control) or presence of 50 µM NOC-18 (NOC-18). (E) Alizarin red dye associated with the cells was dissolved in 10% cetylpyridium chloride, and quantified by measurement of the absorbance at 570 nm after seven- and 14-day cultures with (filled columns) or without NOC-18 (unfilled columns). *Significant difference (n = 4). (F) Alizarin red binding to the cells cultured for 7 days in MP medium in the absence or presence of 5 mM L-NAME. *Significantly lower than the control without L-NAME (n = 4).