Ab174963

Cells and tissues were homogenized for 25 min on ice in RIPA lysis buffer, supplemented with protease inhibitors, phosphatase inhibitors, and 100 mM PMSF (KeyGEN BioTECH, Shanghai, China). Extracted proteins were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany) using a semi-dry apparatus. Membranes were blocked in 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1h at room temperature, followed by overnight incubation with antibodies targeting CXCL12 (1:1000, 3530, Cell Signaling Technology), CXCR4 (1:1000, 11073-1-AP, Proteintech), Wnt5a (1:1000, ab174963, Abcam), β-Catenin (1:5000, ab174963, Abcam), E-cadherin (1:5000, AF1552, Beyotime), N-cadherin (1:2000, AF0243, Beyotime), Vimentin(1:5000, AF0218, Beyotime) and GAPDH (1:2000, ab181602, Abcam) at 4 °C. After washing three times with TBST for 10 min at room temperature, membranes were incubated with secondary antibodies for 1 h, and washed three times with TBST. Positive bands were visualized using an ECL Plus kit (Beyotime, China).

A Leica paraffin slicer RM2235 (Leica Biosystems, Solms, Germany) was used to cut 4 µm-thick sections from each TMA block. Sections were incubated at 70 °C for 1 h, dewaxed in xylene, and rehydrated using an ethanol gradient. Following antigen retrieval by boiling in citrate buffer for 20 min, sections were incubated in hydrogen peroxide for 10 min to quench endogenous peroxidase. Then, tissues were incubated overnight with primary polyclonal antibodies targeting CXCL12 (1:100, 3530, Cell Signaling Technology, Manassas, USA), Wnt5a (1:200, ab174963, Abcam) and β-Catenin (1:300, ab230216) at 4 °C. PBS was used as a negative control. After washing with PBS, sections were incubated with binding buffer and amplification agent (reagent A, GTVisionTM III Kit supply, Shanghai, China), stained with 3, 3'-diaminobenzidine (DAB, reagent B and C, GTVisionTM III Kit supply, Shanghai, China), and counterstained with hematoxylin. Staining was scored based on the percentage of positively-stained cells in each section (no positive staining or ≤ 5% = 0; 6%-25% = 1; 26%-50% = 2; 51%-75% = 3, and 76%-100% = 4), and the staining intensity (no staining = 0, weak staining = 1, moderate staining = 2 and strong staining = 3) 17 (link). For each case, two random fields were imaged, with one core viewed under high magnification (×200). All slides were analyzed by two pathologists in a blinded manner.

The cells were lysed on ice for 30 min with RIPA lysate (Beyotime Biotechnology, Shanghai, China). The supernatant was collected after centrifugation for 14,000 rpm for 15 min at 4 ℃ in a cryogenic centrifuge. BCA kit (Thermo, Shanghai, China) was used to detect the protein concentration. Moreover, the protein loading was modulated according to the protein quantification results, and the protein samples were electrophoresed by 10% SDS-PAGE. Next, the proteins were transferred to the polyvinylidene fluoride (PVDF) membrane, which was then blocked with 5% skim milk for 2 h. After membranes were washed with TBST buffer, primary anti-WNT5A antibody (ab174963, abcam, 1:1000), anti-Bcl-2 antibody (ab196495, abcam, 1:1000), and anti-Bax antibody (ab53154, abcam, 1:1000) were added into the membrane respectively, and incubated for 8 h at 4 ℃. Then the PVDF membranes were washed with TBST, and were incubated at room temperature for 1 h with the secondary antibody. After the membranes were rinsed again, color rendering was performed using hypersensitive ECL (Hubei Biossci Biotechnology, Wuhan, China). The experiment was repeated three times independently.