Figure 3.
PDL-POSTN induces PDL cell differentiation more strongly than the full-length isoform of periostin. (A) PDL-POSTN enhances ALPase activities. MPDL22 cells transfected with control vector, the Type I isoform of periostin, or PDL-POSTN were cultured in mineralization-inducing medium, and ALPase activities were measured on every third day of culture. (B) PDL-POSTN enhanced calcified nodule formation. Alizarin red staining was performed on day 12. The image illustrates the alizarin red staining in each group, with the staining densities quantified in the graph below. (C) PDL-POSTN did not influence proliferation activity. [3H]-thymidine incorporation into transfected MPDL22 cells after stimulation with 10% FCS is shown. Values are the mean ± SD of triplicate assays. (D) Confirmation of stable knockdown of periostin at the mRNA level by real-time PCR analysis (left) and at the protein level by Western blot analysis of the culture supernatants (right). Periostin shRNA-transfected MPDL22 cells cultured in mineralization-inducing medium were analyzed on day 9. (E) Stable knockdown of periostin decreased ALPase activities. Control, MPDL22 cells transiently transfected with a control vector; Type I, MPDL22 cells transiently transfected with a vector expressing periostin type I isoform; PDL-POSTN, MPDL22 cells transiently transfected with a vector expressing PDL-POSTN; shRNA-1, MPDL22 cells stably transfected with mouse periostin siRNA-1 sequences; shRNA-2, MPDL22 cells stably transfected with mouse periostin siRNA-2 sequences; shControl, MPDL22 cells stably transfected with control siRNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. This figure is available in color online at http://jdr.sagepub.com.