Ab254973

PTC samples, along with paired adjacent normal thyroid specimens, were collected from the Affiliated Hospital of Jining Medical University for tissue microarray (TMA) construction. IHC staining of AEBP1 (1: 100, ab254973, Abcam, UK) and BMP4 (1: 200, ab124715, Abcam, UK) using TMA were performed following a standard IHC protocol as described previously [22] (link). The IHC-score (0–12) was obtained by multiplying the score of staining intensity with the score of positive cell frequency [23] (link). The score of staining intensity were defined as: 0 = negative; 1 = weak; 2 = moderate; and 3 = strong. The score of positive cell frequency was defined as: <5 % = 0; 5 %-25 % = 1; 26 %–50 % = 2; 51 %-75 % = 3; >75 % = 4.

All animal procedures and protocols in our study were in accordance with the Institute of Biophysics, Chinese Academy of Science's Policy on Care and Use of Laboratory Animals. Female BALB/c nude mice (5 weeks old) were purchased from the HFK BIOSCIENCE (Beijing). 5 × 10⁶ BCPAP cells with sgAEBP1 or control vector cells in 200 ul PBS were subcutaneously injected into the right flank of mice (6 mice/group). Tumor volumes were measured every 7 days and calculated according to the formula as follows: volume = 0.5 × tumor length × width². Tumor weights were obtained after dissecting the tumor tissues from euthanized mice. The tumor tissues were fixed and dissected for IHC staining with anti-AEBP1 (1: 100, ab254973, Abcam, UK), anti-BMP4 (1: 200, ab124715, Abcam, UK), anti-E-Cad (1: 1000, 14472, CST, USA), and anti-N-cad (1: 200, 13116, CST, USA). For the nude mouse model of pulmonary metastasis assay, PTC cells (1  ×  10⁶) were suspended in 200 µL PBS and injected into mice via the tail vein (5 mice/group). Four weeks after injection, the mice were euthanized, and the lung of dead mice was excised and fixed in formalin. Paraffin-embedded lungs were systematically sectioned and stained with hematoxylin and eosin (H&E) staining, and images were captured by iViewer software.