Fig. 4

Magnolin inhibits epithelial-to-mesenchymal transition. a Effects of magnolin on the protein levels of epithelial-mesenchymal marker proteins. A549 cells were seeded, cultured, treated with the indicated doses of magnolin for 24 h. The proteins were extracted and visualized by Western blotting using the specific antibodies as indicated. b Effects of magnolin on the N- and E-cadherins by immunocytofluorescence analysis in A549 cells. The protein levels of N- and E-cadherins altered by magnolin treatment were visualized by immunocytofluorescence as described in “Materials and Methods”. c RSK2 knockdown effects on the protein levels of EMT marker proteins. Graph, knockdown of RSK2 inhibits cell migration in A549 cells. A549 cells stably expressing sh-mock or sh-RSK2 were seeded into culture-inserts and treated with mitomycin-C for 2 h. The culture-inserts were removed, and the cell migration was measured 24 h later using the Image J computer software program (v. 1.45). Panels, A549 cells stably expressing sh-mock or sh-RSK2 were seeded, cultured and harvested. The protein levels were visualized by Western blotting using the specific antibodies as indicated. d RSK2 deficiency suppresses cell migration and EMT marker protein levels. Graph, RSK2^+/+ mesenchymal and RSK2^−/− MEFs were seeded into culture-inserts and treated with mitomycin-C for 2 h. The culture-inserts were removed, treated with 10 ng/ml EGF and cell migration was allowed for 24 h. The cell migration was observed and measured using the Image J computer software program (v. 1.45). Panels, RSK2^+/+ and RSK2^−/− MEFs were seeded, cultured and harvested. The proteins were extracted and visualized by Western blotting using the specific antibodies as indicated. a, c and d β-actin was used as an internal control to verify equal protein loading. Data are presented as the mean ± S.D. of values from triplicate experiments, and statistical significance was determined using the Student’s t-test (*, p < 0.05)