dgd1364-sup-0001-FigS1.pdfapplication/PDF513K Fig. S1. (A) Westernblot was performed on homogenous palatal MES cells that were treated with small hairpin RNA (shRNA) Smad4 (pRetrosuper-shSmad4 (Addgene, MA, USA) for 48 h to inhibit the Smad-dependent pathway), was used which specifically targets the coding region of murine Smad4. Empty vector and scrambled shRNA vector (scr) were also used as controls. (B) To abolish the effect of endogenous TGFβ3, cells were treated with blocking TGFβ3 antibody was used at 10 ng/mL for 24 h. MES cells were treated with a Smad-independent pathways were blocked using small synthetic chemical inhibitors for 60 min – (C) U0126 for MEK1/2 (20 μmol/L), (D) SB202190 for p38MAPK (20 μmol/L) and (E) LY294002 to block PI3 Kinase (20 μmol/L). (F) All treatment showed significant loss of the targeted protein. Intensity of the band was measured using the Carestream Molecular Imaging Software version 5.3.1 (Rochester). To perform a t-test analysis of mean intensity measurements, a ROI analysis was done from the data to Microsoft Excel software from the exported “.txt” files. (G) In double staining, Keratin 14 (K14) (Rhodamine) was expressed in untreated (i) as well as 24 (ii) and 48 h (iii) of TGFβ3 (5 ng/mL) treatments while Vimentin (FITC; iv), confirm its mesenchynal transformation at 48 h.
dgd1364-sup-0002-FigS2.pdfapplication/PDF513K Fig. S2. Double staining immunofluorescence protein expression assays were performed on homogenous MES cells that were treated with TGFβ3 (5 ng/mL) for 24 and 48 h. In double staining, E-Cadherin (A, FITC and D, Rhodamine) and Desmoplakin (G, Rhodamine) were highly expressed while Vimentin (A, Rhodamine), N-Cadherin (D, FITC) and Fibronectin (G, FITC) expression were not seen in untreated control cells. As expression of Vimentin, Fibronectin and N-Cadherin began to increase, in response TGFβ3 for 24 h (B, E & H), E-Cadherin and Desmoplakin expressions were reduced and significantly lost by 48 h (C, F & I), at which stage, Vimentin, Fibronectin and N-Cadherin were highly expressed.
dgd1364-sup-0003-TableS1.docxWord document14K Table S1. (A) RT-PCR; (B) Chromatin immunoprecipitation (ChIP) assay; (C) Site-directed mutagenesis.

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