E cadherin

Tumor-bearing brain tissue were collected from mice of each group. Tissues were fixed in 10% buffered formalin for 24 h following standard procedure for processing, paraffin-embedding, and sectioning to assess PCNA (1:200, Servicebio, GB11010), E-cadherin (1:200, Servicebio, GB14076) by IHC assay. The reaction was visualized using the Servicebio image analysis system, the staining was scored by two independent and experienced pathologists and calculated as the product of the staining intensity. First divides the positive grade: negative without staining, score 0, weak positive light yellow, score 1, medium positive brown, score 2, strong positive brown, score 3 points. Then analyze and calculate the area of weak, medium, and strong positive in the measurement area, the tissue area of the measurement area, the cumulative optical density value of the positive and the positive area. PCNA and E-cadherin quantification for each sample was determined and molecular data using modified H-scores ([{% of weak staining} × 1] + [{% of moderate staining} × 2] + [{% of strong staining} × 3]), to determine the overall percentage of PCNA and E-cad positivity across the entire stained sample, yielding a range from 0 to 300 [22 (link), 23 (link)].

Western blotting was performed according to standard methods, as described previously [21 (link)]. The tumor sections in brain tissue were snap-frozen in liquid nitrogen for protein isolation, and then tumors were homogenized with a mortar and pestle and lysed in RIPA buffer containing Halt protease and phosphatase inhibitor cocktail. Soluble proteins were quantified by a BCA protein level detection kit and then subjected to SDS-PAGE followed by immunoblotting. Antibody incubation was conducted overnight at 4 °C. Antibodies included EGFR (1:1000, Servicebio, GB11084-2), AKT (1:1000, Servicebio, GB11689), S473 p-AKT (1:1000, Affinity, AF0832), ERK (1/2) (1:1000, Servicebio, GB11560), p-ERK (1/2) (1:1000, Cell Signaling Technology, #4370), E-cadherin (1:1000, Servicebio, GB14076), HIF-1α (1:1000, Servicebio, GB111339), ACTIN (1:3000, Servicebio, GB12001). Secondary antibodies were diluted 1:5000, and immunoreactive proteins were visualized using a Western blotting machine (Thermo) and analyzed with ImageJ software.

Total proteins were extracted and separated by 10% SDS-polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane (Millipore, Burlington, MA, USA). The membrane was incubated in 5% low-fat milk in Tris-buffered saline with 0.1% Tween-20. Subsequently, the membranes were incubated with the rabbit antibodies against human E-cadherin, N-cadherin, vimentin, p-AKT, AKT, p-ERK, ERK, PI3K, and GAPDH (Servicebio, Wuhan, China) at 4 °C overnight and were then incubated with secondary antibodies at 1:5000 dilution. Finally, signals were visualized by an enhanced ECL kit (Millipore, Burlington, MA, USA) (original western blot see Figures S7 and S8).