Supplementary Material for: Function of Transforming growth factor-β2 and -β3 in palatogenesis

Abstract Introduction: This study aimed to examine the transforming growth factor (TGF)-β signaling pathway during secondary palate fusion by transfecting single and double small interfering RNA (siRNAs) for TGF-β2 and -β3. This investigation also focused on understanding the phenotype of palatal development. Methods: siRNAs targeting TGF-β2 and -β3 were used in an organ culture model of fusion of the secondary palate of 13-day embryonic ICR mice cultured for up to 72 h. The palatal shelves were collected at different times following the initiation of organ culture and were examined for TGF-β2 and -β3 gene expression. Downstream signaling was characterized using western blotting and PCR. Results: In the double siRNA-treated palatal shelves, approximately 90% (91% anterior, 89% posterior with phenotype A) showed fusion failure in hematoxylin and eosin staining. Phosphorylation of Smad-dependent and -independent signaling showed a significant reduction in phosphorylation in double knockdown palate organ cultures when compared to single knockdown cultures. Although, the expression of MMP13 and TIMP2 were small influenced by siTGF-β2, the extracellular matrix and transcription factor expressions showed to be significantly reduced in double knockdown palate compared to single knockdown palates. Conclusions: This study demonstrates that double siRNAs targeting TGF-β2 and -β3 results in phenotypes during secondary palatal fusion and that they could be affected phosphorylation of Smad- dependent and -independent signaling synergistically compared to single knockdown of TGF-β2 and -β3. The results of this study demonstrate important functions during secondary palatal fusion and will contribute to our understanding of the etiology of cleft palate.