Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG)
Fig 5
EGCG suppresses EGF-induced colony formation mediated through RSK2 in JB6 Cl41 cells.
(A) EGCG suppresses EGF-induced anchorage-independent growth of JB6 Cl41 cells. JB6 Cl41 cells (8 × 103/mL) were exposed to different doses of EGCG in 1 mL of 0.3% BME agar containing 10% FBS with or without 10 ng/mL EGF. Each dose was repeated in triplicate wells. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days and then colonies were counted using a microscope and the Image-Pro PLUS (vs. 4) computer software program. Data are shown as mean values ± S.D. obtained from triplicate experiments. Differences were evaluated using the Student's t test and the asterisks (*, **) indicate avsignificant inhibitory effect of EGCG on colony formation (p < 0.05; p < 0.01, respectively). (B) EGCG inhibits histone H3 phosphorylation at Ser10. JB6 Cl41cells (5 X 105) were seeded into 10-cm dishes in 5% FBS/MEM and cultured until cells reached 80–90% confluence. The cells were starved for 24 h in 0.1% FBS/MEM. Cells were treated with the indicated dose of EGCG or kaempferol (as positive control) for 1 h and then stimulated with EGF (10 ng/mL) for 15 min and subsequently harvested. Histone proteins were extracted as described in Materials and Methods. The phosphorylation of histone H3 (Ser10) was detected by Western blot. Equal protein loading and transfer were confirmed by stripping and incubating the same membrane with an antibody against total histone H3. Each assay was performed three times and similar results were obtained. Representative blots are shown.
