Figure 2

Under conditions of proteoglycan sulfation inhibition, alkaline phosphatase activity is present in mononuclear skeletal muscle cells. A. C2C12 cells were induced to differentiate for 6 days in the absence (first column) or presence of 30 mM sodium chlorate (second column). Remaining mononuclear cells from control (third column) or sodium chlorate treated cultures (fourth column) were isolated and grown for 48 hours. C2C12 cells induced to differentiate in the presence of 5 nM BMP-2 were used as positive control (fifth column). Unpermeabilized cells were fixed and alkaline phosphatase activity was visualized as a fluorescent precipitate using ELF-97 detection kit; nuclear staining was performed with 1 μg/ml Hoechst 33258 (lower row). Phase contrast microscopy is shown in the upper row. Bar = 25 μm. B. Percentage of ALP-positive mononuclear cells in the different experimental conditions described in A. Values correspond to mean ± S.E.M. of 10 different microscopy fields from two independent experiments. ANOVA analysis followed by Tukey-Kramer multiple comparisons test shows there are no statistically significant differences when both control or both chlorate conditions (p > 0.05) are compared. All other comparisons are significantly different (p < 0.001).