β-TRCP regulates VEGFR2 protein levels in human poorly differentiated PTC-derived cells. (A and B) Immunoblot analysis of various human thyroid cancer cell lines with indicated antibodies. Data are representative of two independent experiments. (C) Human PTC with squamous metaplasia (SM) were stained with the indicated antibodies and analyzed by immunohistochemistry. Low β-TRCP is defined as 1–10% of endothelial or human PTC cells with SM foci being positive for β-TRCP staining. High β-TRCP is defined as >50% of endothelial or human PTC cells with SM being positive for β-TRCP. H+E, H&E staining. Bars: (400×) 200 µm; (1,000×) 50 µm. Arrows and arrowheads highlight endothelium and tumor cells, respectively. (D) Quantitative measurement of the microvascular density (as defined by number of vessels per field showing CD31 staining in PTC samples expressing low or high amounts of β-TRCP1) shown in C. The error bars represent mean ± SD. ***, P < 0.001 (n = 3). (E and F) Immunoblot analysis of BCPAP cells infected with either EV or HA–β-TRCP1-encoding lentiviral vectors. Where indicated, cells were treated with the proteasome inhibitor MG132. Data are representative of two independent experiments. (G and H) Real-time RT-PCR analysis of VEGFR2 (G) and β-TRCP1 (H) mRNA in BCPAP cells infected with EV or HA-β-TRCP1–encoding lentiviral vectors. Data are representative of three independent experiments. The error bars represent mean ± SD. ***, P < 0.001.