Fig. 5

ECM stiffness acts via RAP1a/ARHGAP35 to regulate YAP in ESCs. a Pull-down assays of total Rap1a and activated Rap1a (Rap1a-GTP) in ESCs plated on 1 kPa and 40 kPa hydrogels for 24 h. b Western blotting for YAP in nuclear and cytoplasmic protein fractions from ESCs transfected with si Rap1a and plated on 1 kPa fibronectin-coated hydrogels for 24 h. c, d Confocal immunofluorescence images (c) and quantification of the nuclear and cytoplasmic subcellular localization (d) of YAP in ESCs plated on 1 kPa hydrogels and transfected with si Rap1a. Scale bar: 20 μm. e Coimmunoprecipitation (IP) of p190A and Rap1a in ESCs plated on 1 kPa hydrogels. Cell lysates were immunoprecipitated with Rap1a antibodies or with the corresponding IgG controls and probed with the indicated antibodies. f, g Confocal immunofluorescence images (f) and quantification of the average fluorescence intensity (g) of p190A in ESCs. Cells were plated on 1 kPa fibronectin-coated hydrogels and transfected with si Rap1a or si NC for 24 h. Scale bar: 10 μm. h–j. ESCs were plated on 40 kPa fibronectin-coated hydrogels and transfected with pcDNA3.1+ Rap1a or si ARHGAP35 or pcDNA3.1+ Rap1a and si ARHGAP35 or pcDNA3.1+ NC for 24 h. h, i. Confocal immunofluorescence images (h) and quantification of the nuclear and cytoplasmic subcellular localization (i) of YAP in ESCs. Scale bar: 10 μm. j, k Total cell lysates or nuclear and cytoplasmic lysates of ESCs were prepared and subjected to immunoblot analysis with the indicated antibodies. l RT‒qPCR analysis of CYR61, CTGF and ANKRD1 mRNA levels in ESCs. Data are means ± S.E.M. of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001