Transgelin

All specimens were fixed in 10% neutral formaldehyde. Paraffin embedded, 4 μm continuous section. Xylene dewaxing, gradient ethanol hydration. Antigen thermal repair with 0.01 mol/L sodium citrate buffer. Endogenous peroxidase was sealed at 37°C for 20 min. Primary antibody (Transgelin, 1:200, Abcam; CD31, 1:1000, Abcam) was added and incubated overnight at 4°C. The secondary antibody was added and incubated at 37°C for 30 min. Horseradish peroxidase was added and incubated at 37°C for 20 min. DAB staining, hematoxylin redyeing, gradient ethanol dehydration. Xylene transparent, neutral gum sealed sheet, light microscope detection and photography. The staining results were graded according to the staining intensity and percentage of positive cells. The percentages of positive cells <5%, 5%–25%, 26%–50%, 51%–75%, and >75% were 0, 1, 2, 3, and 4, respectively. The cell staining intensity was scored as follows: 0 for non-staining, 1 for light yellow, 2 for brownish yellow, and 3 for yellowish brown. The positive intensity is the product of two scores: 0–2 is negative, 3–5 is weakly positive, 6–8 is positive, and 9–12 is strongly positive. The results were determined by two pathologists, and the average value was taken.

Cells were lysed in RIPA buffer, and protein concentration was measured using DC Protein Assay Kit (BioRad). Proteins of 20–30 μg were run on the SDS-PAGE gel, and the expression of target proteins were assessed by immunoblotting. Target protein expression levels were normalized to GAPDH expression levels. Primary antibodies used were as follows: FAM13A (Sigma, HPA038108), transgelin (TAGLN, Abcam, #ab14106), Snail (Cell Signaling Technology, C15D3), PECAM-1 (Santa Cruz, #sc-1506), caspase-3 (Cell Signaling Technology, 8G10), cleaved caspase-3 (Cell Signaling Technology, D175), β-catenin (Cell Signaling Technology, #9562), active β-catenin (Cell Signaling Technology, D13A1), GAPDH (Cell Signaling Technology, 14C10). All antibodies were used at 1:1000 dilution unless otherwise mentioned.