Fig. 5. Effects of kinase inhibitors on berberine-induced NAG-1 expression in HCT-116 cells.

(A) HCT-116 cells were treated with DMSO, PD98059 (40 μM), AKT inhibitor (10 μM), MG132 (10 μM), RO31-8220 (2.5 μM), AR-A014418 (30 μM), AG490 (50 μM) or SP600125 (30 μM), for 30 min followed by treatment with 50 μM of berberine for an additional 24 h in the absence of serum. The cell lysates were harvested, and 30 μg of total protein were subjected to Western blot analysis as described in Materials and Methods. Equal amounts of loading proteins were estimated by Actin probing. (B) HCT-116 cells were treated with DMSO, RO31-8220 (1, 2.5 and 5 μM), Rottlerin (0.5, 1 and 5 μM) and Go6983 (10, 50 and 100 nM) in serum-free media. After 30 min, either DMSO or 50 μM of berberine was added directly to the media. After 24 h, cells were both harvested and analyzed by Western blot, as described in Materials and Methods. (C) HCT-116 cells were serum starved for 24 h and treated with 50 μM of berberine. At the indicated times, the cell lysates were harvested to perform Western blot analysis for p-ERK1/2, total ERK1/2, and NAG-1, and results were normamlized with Actin. (D) The pNAG133/+41 luciferase construct (0.5 μg) was transiently transfected with 0.05 μg of pRL-null vector into HCT-116 cells using lipofectamine. The cells were pretreated for 30 min in serum free media with DMSO, PD98059 (40 μM), AKT inhibitor (10 μM), MG132 (10 μM), RO31-8220 (5 μM), AR-A014418 (30 μM), AG490 (50 μM) or SP600125 (30 μM), and 50 μM of berberine was added in all wells except vehicle. After 24 h, luciferase activity was measured. The y axis shows fold increase in relative luciferase units (RLU) compared to RLU of vehicle-treated cells. Each value represents mean ± SD from three independent transfections. The student t- test was used to determine significance in the NAG-1 promoter activity in the presence of berberine, and the RLU of all the different inhibitor treated samples were compared with that of the berberine-only treated sample.