Fig. 4

CDK2 mediates BMP2K function in MLN8237-induced megakaryocytic differentiation. a The luciferase activity of an E2F-responsive element in 293T cells co-transfected without (-) or with an increased amount (triangle) of BMP2K overexpression vector. ***, p < 0.001 for comparison as indicated. b Control and BMP2K overexpression cells were treated with Nocodazole (10 ng/ml) for 6 h followed by wash and culturing in fresh medium for 3 or 5 h as indicated (Nocodazole Withdrawal). The cell cycle profile was measured by DAPI staining followed by flow cytometry analysis. c The presence of BMP2K in the nuclear extract (N) from BMP2K overexpression cells was detected by immunoblotting. Lamin A and HSC70 serve as positive proteins for nuclear extract and cytosolic extract (c), respectively. d The absence or presence of BMP2K-FLAG and CDK2-HA expression plasmids were indicated as – or +, respectively. Plasmids with different combinations were transfected into 293T cells for co-immunoprecipitation with FLAG antibody (IP: FLAG, left panel) or with HA antibody (IP: HA, right panel). The BMP2K-FLAG and CDK2-HA in IP and lysates (INPUT) was detected by immunoblotting. e–f MLN8237 together with Vehicle or CDK2 inhibitor K03861 (10 nM) as indicated induced Control and BMP2K overexpression cells undergoing megakaryocytic differentiation, which was evaluated by CD41 staining (e) and DAPI staining for DNA content (f). g–h CDK2 overexpression in shBMP2K#2 cells (shBMP2K#2 + CDK2) as indicated offset the effect of BMP2K deficiency on MLN8237-induced megakaryocytic differentiation, which was measured by CD41 straining g and DAPI staining for DNA content h Gates in F and H represent cells with ploidy ≥ 8 N. * p < 0.05, ** p < 0.01; *** p < 0.001; NS, non-significance compared with Control or Scramble cells