Figure 3
Analysis of SMAD1-5-9 and p38 activation upon IdU treatment. Representative western blot at (a) early and (b) late time points of p-SMAD1-5-9 and p-p38. The complete gels are available at Figures S6 and S7 for panel (a) and (b) respectively. (c,d) Densitometric quantitation of bands in panel (a) and (b). Data are represented as fold induction relative to vehicle-treated sample (horizontal line = 1). Statistical significance was assessed by a Two-Way Anova test. **p < 0.01; ***p < 0.001; ****p < 0.0001; n = 3. (e) Representative image of ALP staining of MABs pre-treated with vehicle or 10 μM SB 203580 for 3 hours in growth medium and then treated with 25 μM IdU in combination with 10 μM SB 203580 for 5 days in growth medium. Scale bar 200 μm. (f) ALP positive area quantitation of images in panel (e). Data are presented as percentage of ALP positive area over the total area of the image filed ± SEM. Statistical significance was assessed by a Student’s t-test. *p < 0.05; n = 3. (g,h) Bar plot of real time PCR analysis of Runx2 and Sp7 of MABs pre-treated with vehicle or 10 μM SB 203580 for 3 hours in growth medium and then treated with 25 μM IdU in combination with 10 μM SB 203580 for 48 hours in growth medium. Data are presented as fold change versus untreated sample (horizontal line = 1) ± SEM. Statistical analysis was performed by a Two-Way Anova. “Asterisk” symbol (*) represents the significance versus CTRL sample. *p < 0.05; ****p < 0.0001. “Plus” symbol (+) represents the significance between Vehicle and SB pre-treated sample. ++p < 0.01. n = 3. (i) ALP staining of MABs treated with IdU in combination with BMP-2. MABs were treated and cultivated for 120 hours in growth medium. Osteogenic differentiation was assessed by ALP staining. ALP positive area percentage is reported at the bottom-left side of each micrograph. Red-squared images show an additive effect between BMP-2 and IdU. The complete quantitation of ALP staining is shown in Figure S2.
