Research Paper Volume 11, Issue 17 pp 7051—7069

Effects of age-dependent changes in cell size on endothelial cell proliferation and senescence through YAP1

Figure 3. CDC42-YAP1 mediates cell size-dependent changes in EC senescence in aged ECs. (A) Representative immunoblots showing YAP1, YAP1S127 phosphorylation, and β-actin protein levels in ECs isolated from >50 y.o. human adipose tissues treated with retrovirus overexpressing full-length YAP1 or YAP1S127A (left). Graph showing the quantification of immunoblots (right, n=3, *, p<0.05). (B) IF micrographs showing P16INK4A expression and DAPI in ECs isolated from >50 y.o. human adipose tissues treated with retrovirus overexpressing full-length YAP1 or YAP1S127A, cultured on FN-coated island of different sizes. Scale bar, 10 μm. (C) Graph showing quantification of P16INK4A integrated density (n=7, mean ± s.e.m., *, p<0.05). (D) IF micrographs showing the GTP-CDC42 levels and DAPI in ECs isolated from >50 y.o. human adipose tissues treated with ML141 (500 nM). Scale bar, 10 μm. Graph showing quantification of GTP-CDC42 integrated density (n=7, mean ± s.e.m., *, p<0.05). (E) Representative immunoblots showing YAP1, YAP1S127 phosphorylation, and β-actin protein levels in ECs isolated from >50 y.o. human adipose tissues treated with ML-141 (top). Graph showing the quantification of immunoblots (bottom, n=3, *, p<0.05). (F) IF micrographs showing P16INK4A expression (green) and DAPI (blue, top) and YAP1 localization (green), actin structure (magenta), and DAPI (blue, bottom) in ECs isolated from >50 y.o. human adipose tissues treated with ML141 and cultured on FN-coated island of different sizes. Scale bar, 10 μm. Graphs showing quantification of P16INK4A integrated density (bottom left) and nuclear YAP1 (bottom right) (n=7, mean ± s.e.m., *, p<0.05).