Fig. 2

FK506 induces Lymphatic endothelial cell senescence. Lung lymphatic endothelial cells were treated with FK506 (15 ng/ml) for 72 h. Whole cell lysates were analyzed by Western blotting with antibodies against p21(a) and β-galactosidase (e). Ratio of p21 (b) and β-galactosidase (f) to β-actin density was expressed as fold-change relative to control. Data represent mean ± SEM of three independent experiments (one technical replicate each) * p < 0.05 by one-way ANOVA. Representative images of immunofluorescent staining of single and merged images of p21 (green) and 4′,6-diamidino-2-phenulindole (DAPI; blue) at low and high magnification (far right panel) with and without treatment with FK506 (15 ng/ml) for 72 h (c). Random images (9–12 images) were obtained and nuclear intensity of p21 staining was measured using Image J software (d). Data represent mean ± SEM of one experiment (**p < 0.01). Results were reproduced in 3 independent experiments (Size bar: 40 μm)