Fig. 3

MTX suppresses YAP activation, whereas silencing AMPKα abolishes this effect. a Representative western blots of p-YAP (Ser127), YAP, p-AMPK, total AMPK (AMPK), β-actin, ICAM-1, and VCAM-1 in HUVECs after each treatment. Values represent the mean ± SD from three independent experiments from each group. **p < 0.01, ****p < 0.0001 vs. DF; ^##p < 0.01, ^###p < 0.001 vs. DF + MTX. b Immunofluorescence staining of p-YAP and YAP in HUVECs. p-YAP (green) and YAP (red) were visualised by immunostaining; nuclei were counterstained with propidium iodide (PI, blue). Boxed regions are enlarged images. The graphic data are the YAP intensity ratios (nuclear/cytoplasmic) of cells randomly selected from three independent experiments. **p < 0.01 vs. DF, ^####p < 0.0001 vs. DF + MTX. MTX decreased YAP nuclear localisation under DF; siAMPKα counteracted the effects. c Representative images of THP1 cell (blue) adhesion to the differently-treated HUVECs under DF. The graphic data show quantification of THP1 cells from three independent experiments. d AMPK, as measured by qRT-PCR (n = 3) and western blotting (n = 3), in HUVECs after transfection with siNC or siAMPKα for 36 h. ***p < 0.001, ****p < 0.0001 vs. siNC