Fig. 2.

ROCK2 silencing reduces YAP expression and YAP-mediated transcriptional activity. a Immunofluorescence staining of YAP in U-2OS cells after 24 h of exposure to the Stemolecule™ ROCK2 Inhibitor (10 μM) or siRNA sequences targeting ROCK2 (siROCK2) or irrelevant target sequences (SCR). Digital images were taken under identical conditions using the image analysis software NIS-Elements (Nikon Italia); scale bar, 20 μm. b Western blotting of YAP in cytoplasmic and nuclear fractions of U-2OS cells after 24–48 h of exposure to ROCK2 inhibitor together with densitometric analysis. YAP signal was quantified against GAPDH or LAMIN B and reported as ratio of adjusted volume optical density (OD/mm²). Data are presented as the mean ± standard error (SE) of three separate experiments (** p < 0.01, Student’s t test) c qPCR analysis of the expression of CYR61, CTGF and CCND1 in U-2OS parental cells after 24-h to 72-h treatments. Data are shown as 2-^ΔΔCt. GAPDH was used as a housekeeping gene. Data are presented as the mean ± standard error (SE) of three separate experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t test)