Fig. 2

Involvement of YAP in ABF-induced apoptosis. a Effects of ABF (20 nM) on the expression of YAP in MCF-7 cells. Whole cell lysates were evaluated by Western blotting with β-actin as the loading control. b Quantification of protein levels was analyzed by ImageJ Software. Data represent the mean ± SEM, n = 3. ^***P < 0.001 versus the control. c Quantitative data of the YAP shift induced by ABF. Data represent the mean ± SEM, n = 3, ^***P < 0.001 versus the control. d ABF promotes the nuclear translocation of YAP. Following treatment with ABF (20 nM), the cells were fixed and incubated with the indicated antibodies. Immunofluorescence images were obtained by confocal scanning laser microscopy (LSM 800, Zeiss). Original magnification: 400 × ; Scale bar: 10 μm. e Quantitative results of the relative fluorescence intensity of YAP in the nucleuses. Data represent the mean ± SEM, n = 5, ^**P < 0.01 versus the control. f The level of the YAP/p73 complex in ABF-treated cells. The YAP/p73 complex was pulled down by coimmunoprecipitation with an anti-YAP antibody. Total cell lysates were used as the input control. g Quantification of the YAP/p73 complex in ABF-treated cells. Data represent the mean ± SEM, n = 3, ^*P < 0.05 versus the control. h ABF (20 nM) upregulated the mRNA levels of Bax and p53AIP1. The mRNA levels of Bax and p53AIP1 were determined by quantitative real-time PCR. Each column represents the mean ± SEM (n = 3). ^***P < 0.001 versus the control. i–l YAP siRNA antagonizes ABF-induced apoptosis. Cells were pretreated with YAP siRNA and incubated with ABF (20 nM). Then, treated cells or cell lysates were evaluated by the Annexin V-FITC/PI staining assay (i), cell viability assay (j), and Western blotting (k). l Quantification of the expression of the indicated proteins. Each column represents the mean ± SEM, n = 3. ^***P < 0.001 versus the control; ^###P < 0.001 versus the ABF group