FIGURE 2.

Delayed and decreased mineralization of 17A odontoblastic cells overexpressing Trps1. A, qRT-PCR (top panel) and Western blot (bottom panel) results demonstrating overexpression of Trps1 in three clonal stable cell lines (Trps1-OE) and controls (Cntr, undifferentiated cells). qRT-PCR data are presented as the mean relative levels of Trps1 mRNA normalized to Gapdh ±S.D. from three independent RNA preparations per cell line. Relative Trps1 levels in one of the Cntr analyses were arbitrarily set at 1 and used as a reference for the remaining control and Trps1-OE cell lines. On the Western blot analyses, tubulin was used as a protein loading control. B, representative images of alizarin red staining (top panel) and quantification (bottom panel) of Trps1-OE and Cntr cell lines during osteo-odontogenic differentiation. Quantification of alizarin red staining is presented as the mean ± S.D. from differentiation of three stable cell lines. Asterisks denote statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.005). C, representative images of TNAP activity assay in Trps1-OE and Cntr undifferentiated (day 0) and differentiated (day 9) cells (left panel) and results of densitometric quantification of TNAP activity (right panel). Data are presented as the mean ± S.D. from differentiation of three stable cell lines. No statistically significant difference was detected. D, qRT-PCR results demonstrating dynamic Dspp expression during osteogenic differentiation of Trps1-OE and Cntr cell lines. Data are presented as the mean relative levels of Dspp mRNA (normalized to Gapdh) ±S.D. from three stable cell lines. Dspp expression was not detected in undifferentiated controls; therefore, values for day 1 of Cntr cells were arbitrarily set at 1 and used as a reference for the remaining time points.