Figure 4.

Impaired SMAD1/5 nuclear entry and interaction with SMAD4 upon SMYD2 knockdown or knock-out. A, immunofluorescence (IF) staining analysis showing impaired BMP2-induced nuclear translocation upon knockdown of SMYD2 by siRNA. HaCaT cells were treated with or without siSMYD2-1 for 2 days. The cells were then serum-starved overnight, stimulated without or with BMP2 for 25 min, and processed for immunofluorescent staining analysis using an anti-SMAD1 antibody. B, immunofluorescence staining analysis showing impaired BMP2-induced nuclear translocation upon knock-out of SMYD2. The SMYD2 KO-16 cells were serum-starved overnight, stimulated without or with BMP2 for 25 min, and processed for immunofluorescent staining analysis. C, impairment of BMP2-induced interaction between SMAD1 and SMAD4 upon knock-out of SMYD2. Control cells and SMYD2 KO-16 cells were serum-starved overnight and stimulated with BMP2 for 40 min. The cells were harvested, and co-immunoprecipitation assay was performed using anti-SMAD1 antibody followed by Western blot analysis using anti-SMAD4 and anti-SMAD1 antibodies. D, SMYD2 knock-out diminished cell proliferation inhibition by BMP2. The wild-type and SMYD2 KO-16 cells were treated without or with 25 ng/ml BMP2 for up to 96 h, and cell proliferation was assayed by CCK8 assay at 0, 24, 48, 72, and 96 h as indicated.