Immunohistochemical staining was performed as previously described [20 (link)]. The negative control was detected with the primary antibody replaced by PBS. Immunohistochemical staining was scored by two independent observers who were blinded to the patients’ clinical data. The scoring system for grading the level of YAP was reported previously [22 (link)]. The expression level was evaluated by immunohistochemistry (IHC) score calculated by multiplying a proportion score and intensity score. The proportion score reflected the fraction of positive-stained cells (0, none; 1, ≤ 10%; 2, 10–25%; 3, 25–50%; and 4, > 50%), and the intensity score revealed the staining intensity (0, no staining; 1, weak; 2, intermediate; and 3, strong). Finally, the total score was calculated. High and low protein expression levels were defined using the mean score of all samples as a cutoff point. With these criteria, tissue staining could be interpreted as “low” or “high.” The primary antibodies used were anti-YAP (Proteintech Group, 13584-1-AP, IL, USA), anti-Ki67 (Proteintech Group, 27309-1-AP), and anti-c-myc (Abcam, ab32072, Cambridge, UK). For hematoxylin-eosin (H&E) staining, fresh subcutaneous tumors isolated from mice were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin for histological examinations. Sections with thickness of 4 μm were cut and stained with H&E.
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