Areca nut induces TGF-β signaling in epithelial cells.

<p>A & B] Areca nut induced genes by Western blot and by qRT-PCR. HaCaT cells were serum starved for 24 hours and treated with either areca nut water (H) or ethanol (Et) extracts 2.5, 5 µg/ml (2.5H/5H & 2.5Et/5Et) in serum free medium for 48 hours. A) Western blot of HaCaT cell lysates showing induction of p-SMAD2 and its downstream target TGM2. B) qRT-PCR analysis showing expression of TGF-β down-stream target genes TGM2, TMEPAI, THBS1 and TGFBI. <b>UN</b>-Untreated, 2.5H & 5H− 2.5 and 5 µg/ml Areca nut water extract, 2.5 Et,& 5Et- 2.5 and 5 µg/ml, <b>T</b>-TGF-β (_*** = P<0.0001 compared to untreated). C & D] ALK5 inhibitor (SB 431542) reverses the Areca nut induced expression of genes in HaCaT cells. HaCaT cells were treated with areca nut water (H) and ethanol (Et) extracts (5H− 5 µg/ml areca nut water extract and 5Et- 5 µg/ml areca nut ethanol extract) in the presence or absence of ALK5 (TβRI) inhibitor. Western blot of p-SMAD2 and TGM2 (C). qRT-PCR of TGM2,TMEPAI, THBS1 and TGFBI (D). Untreated, 5H− Areca nut water extract (5 µg/ml), 5Et- Areca nut Ethanol extract (5 µg/ml), <b>ALK5</b>- TβRI inhibitor, 5H+ALK5 inhibitor, 5Et+ALK5 inhibitor (_*** = P≤0.0001 compared to untreated).