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. 2010 Jul 19;31(8):944–952. doi: 10.1038/aps.2010.72

Figure 5.

Figure 5

Withagulatin A inhibits TGF-β stimulated procollagen I mRNA expression and Smad2/3 phosphorylation. (A, B) Withagulatin A inhibits TGF-β stimulated procollagen I mRNA expression. (A) Primary rat HSCs and (B) LX-2 cells were treated with Withagulatin A (0, 1, and 2 μmol/L) in the presence of TGF-β (2 ng/mL) for 24 h in DMEM supplemented with 0.2% BSA. mRNA levels of α1 procollagen I and α2 procollagen I were analyzed by real-time PCR assays. Ribosomal 18s RNA was used as an internal control. bP<0.05, cP<0.01 compared with DMSO treated cells. (C) Withagulatin A suppresses TGF-β stimulated Smad2/3 phosphorylation. Primary rat HSCs were treated with Withagulatin A (0, 1, and 2 μmol/L) in the presence of TGF-β (2 ng/mL) for 24 h in DMEM supplemented with 0.2% BSA. Cells were harvested and subjected to Western blot analysis for phosphorylated Smad2 (Ser 465/467), Smad2, phosphorylated Smad3 (Ser 432/425), and Smad3. GAPDH was used as a loading control. Results shown are representative of three independent experiments. (D) Bands were quantified and data are expressed as fold of control. (E, F) Withagulatin A suppresses TGF-β induced Smad2 nuclear translocation. CHO/EGFP-Smad2 cells were pretreated with Withagulatin A (0, 1 and 2 μmol/L) for 10 h and then stimulated with TGF-β (2 ng/mL) for 2 h in serum free F-12 medium supplemented with 0.2% BSA. Finally, cells were stained with 2 μmol/L Hoechst 33342 for 15 min and fluorescent images were taken by an INCell Analyzer 1000. Each treatment was repeated in 3 wells and 5 fields were photographed for each well. (E) Representative views are presented (scale bar=50 μm) and (F) data were quantified using the INCell Analyzer analysis software. cP<0.01 vs TGF-β stimulated cells.