Fig. 1
Silencing ROCK2 in OS cells impairs tumor growth and metastasis in mice and downregulates YAP expression and activity. a Tumor volume and b representative images of lung metastases of mice injected subcutaneously with U-2OS cells modified for ROCK2 expression. n = 6 animals per group. *p < 0.05, **p < 0.001 vs. control (U-2/SCR pool) by the Student’s t test. c ROCK2 and YAP expression were evaluated by the immunostaining of paraffin-embedded tissue samples. Representative images from one tumor developed after injection of control (U-2/SCR) or silenced cells (U-2/shROCK2#78). Scale bar, 50 μm; 200x of magnification with zoomed insert to show details. d Western blotting of snap-frozen tissue samples from two representative tumors that formed in mice injected with control (U-2/SCR) or silenced cells (U-2/shROCK2#78). GAPDH was used as a loading control. ROCK2 or YAP signals were quantified against GAPDH and reported as ratio of adjusted volume optical density (OD/mm2). e YAP activity was evaluated by measuring the relative mRNA expression of its downstream targets CTGF and CCND1 by qPCR. Scatter plot analysis of their expression in the control and U-2shROCK2#78-derived xenografts (n = 6) is shown. The 2−ΔCT method, where ΔCT = CT target gene – CT GAPDH, was used. Bars represent mean ± SE, *p < 0.05, ***p < 0.001, Student’s t test