Figure 6. Curcumin downregulation the expression of Snail by supressing Smad2 phosphorylation and nuclear translocation.

(A) Cells were pretreated with or without 20 μM curcumin for 4 h and then treated with or without 20 ng/ml TGF-β1 for 10 min, 15 min, 30 min respectively. The phosphorylation of Smad2 and total Smad2 were detected by western blotting. GAPDH served as the loading control. Similar results were obtained in three independent experiments. (B) Cells were pretreated with or without 20 μM curcumin for 4 h and then treated with or without 20 ng/ml TGF-β1 for 30min, immunofluorescence and confocal microscopy detection of Smad2 were performed as described in Section 2. (C) Cells were pretreated with or without 20 μM curcumin for 4 h and then treated with or without 20 ng/ml TGF-β1 for 1 h, the combination of Smads and Snail promoter was detected by EMSA. (D) Cells were transfected with pGL3-Basic-Snail-luc reporter plasmid for 6 h, then cells were pretreated with or without 20 μM curcumin for 4 h and then treated with or without 20 ng/ml TGF-β1 for 24 h. Luminescence was measured by a luminometer. pRL-TK plasmids served as the correcting transfection efficiency. Results were expressed as the ratios between the activity of the reporter plasmid and pRL-TK. Similar results were obtained in three independent experiments. Statistically significant values with P < 0.01 are marked with (^*).