Ab124715

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.

Total protein was lysed with RIPA buffer (KeyGEN, Biotech), and the protein concentration was determined using a BCA Protein Assay Kit (ZJ101, EpiZyme). Total protein was separated by SDS‒PAGE gel electrophoresis and transferred onto PVDF membrane (Millipore, USA). Membranes were blocked in 5 % skim milk for 1 h and incubated overnight with primary antibodies at 4°C. Then, membranes were incubated with secondary antibody and visualized. The primary antibodies used were anti-AEBP1 (1: 1000, sc-271374, Santa Zruz, USA), anti-GAPDH (1: 10000, AC003, Abclonal, China), anti-BMP4 (1: 2000, ab124715, Abcam, UK), anti-E-cad (1: 2000, 14472, CST, USA), anti-N-cad (1: 1000, 13116, CST, USA), anti-vimentin (1: 1000, 3932, CST, USA), anti-TWIST1 (1: 1000, 90445, CST, USA), anti-ZEB2 (1: 1000, ab223688, Abcam, UK), anti-Smad1/5/9 (1: 1000, ab300164, Abcam, UK), and anti-p-Smad1/5/9 (1: 1000, AP0850, Abclonal).

Briefly, three-micron thick paraffin sections were applied to silane-coated glass slides, left overnight to dry in an incubator at 60°C, then deparaffinized in xylene, and rehydrated in graded concentrations of alcohol and distilled water. Samples were then incubated at room temperature with 0.3% hydrogen peroxidase (H₂O₂) in methanol for 10 minutes to block endogenous peroxidase activity. Antigen retrieval was performed in a water bath in Tris-EDTA buffer (pH 9.0) at 95-97°C for 15 minutes, and then treated with anti-BMP-4, rabbit anti-human monoclonal antibody (Abcam/code ab124715; dilutions 1:400) with prior antigen retrieval in Tris-EDTA buffer (pH 9.0) in a water bath for 15 minutes at a temperature of 97 °C. Immunohistochemical evaluation of BMP-4 expression was classified as negative, weak diffuse, moderate diffuse and strong granular cytoplasmic staining. For each staining pattern it was determined on the majority of the sample (more than 75% of cells), except for samples with at least 10% of cells showing granular staining pattern, which was characterized as strong granular staining (7 (link)).
Analysis of BMP-4 expression was conducted according to presence/absence of expression and type of expression in a group of positive tumors, as non-granular (weak and moderate diffuse positivity) and granular (strong granular positivity).

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.