Sall4

Fresh tissues were fixed with formalin and paraffin embedded. All the tissue samples were cut into 4 μm sections. After deparaffinization and rehydration, sections were boiled in citrate buffer at 100 °C for 3 min and immersed in 3% H₂O₂ for 10 min for endogenous peroxidase blocking. First, the sections were incubated overnight with the antibody (Ki-67, 1:20, Millipore; SALL4, 1:500, Abcam; DNMT1, 1:200, CST; DNMT3b, 1:500, CST; VEGF, 1:500, CST; MMP-9, 1:300, CST). Then, washed with PBS and incubated in goat anti-rabbit IgG HRP (CST, Darmstadt, DE) for 30 min. Finally, stained with DAB (Solarbio, Beijing, China) and then analyzed the stained cells. Five fields were randomly selected. The protein IHC expression in tissues was normalized by IHC scores, according the method described previously [38 (link)].

Total protein was extracted following lysis in RIPA buffer (25 mM Trizma pH 7.4, 150 mM NaCl, 0.1% SDS, .5% sodium deoxycholate, and 1% NP-40 substitute (Sigma, 74385). Protease inhibitors were added to RIPA as follows: 1:1000 DTT (Sigma, 646563), 5:1000 PMSF (Sigma, 93482-50ML-F), and 1:1000 Protease Inhibitor Cocktail (Sigma, P8340). 10 μg of protein was run for each sample on a 4–20% Criterion Tris-HCl protein gel (Bio-Rad 3450033) and processed using standard western blot technique. All primary incubations were performed overnight in 5% BSA/TBST. Secondary incubations were done in 5% milk/TBST for approximately 1 h. Imaging was performed on a GE Amersham Imager 600. Analysis was done in ImageJ and values were normalized to GAPDH. Western Blot: Sall4 (Abcam, ab29112), GAPDH (Santa Cruz, sc25778), Nanog (Santa Cruz sc8822), Oct3/4 (Santa Cruz, sc9081), Tet1 (Abcam, ab191698), Tet2 (Abcam, ab94580), donkey anti-rabbit IgG-HRP (Santa Cruz, sc2313), m-IgGκ BP-HRP (Santa Cruz, sc516102), Brachyury/T (Santa Cruz, N-19 sc17743).

Cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 6.8, 100 mM 2-ME, 2 %w/v SDS, 10 % glycerol). After centrifugation at 20,000×g for 10 min at 4 °C, proteins in the supernatants were quantified and separated with 10% SDS-PAGE. Then, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Bioscience, Buckinghamshire, USA), which was then incubated with PBS containing 5% milk overnight at 4 °C. The PVDF membrane was incubated with rabbit anti-human primary antibodies including SALL4 (polyclonal, 1:100, ab29112), E-cadherin (polyclonal, 1:50, ab15148), N-cadherin (polyclonal, 1:50, ab18203), Fibronectin (polyclonal, 1:200, ab2413), vimentin (monoclonal, 1:50, ab16700), MMP2 (polyclonal, 1:200, ab37150), MMP9 (polyclonal, 1:100, ab38898), and GAPDH (polyclonal, 1:100, ab181602) (all from Abcam, Cambridge, MA, USA) at room temperature for 3 h, respectively, and then with mouse anti-rabbit secondary antibody (monoclonal, 1:10000, ab99702, Abcam) at room temperature for 1 h. Super Signal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, USA) was then used to detect signals, according to the manufacture's instruction. The relative protein expression was analyzed by Image-Pro plus software 6.0, represented as the density ratio versus GAPDH.

Immunohistochemistry staining was carried out as we previously described [16 (link)]. Morphology analysis of formalin-fixed and paraffin-embedded (FFPE) sections were performed with hematoxylin and eosin staining. For IHC staining, heat-based antigen unmasking was conducted using microwave in citrate buffer after dewax and rehydration of sections. Tissue sections were exposed to 3% H₂O₂ to block endogenous peroxidase, followed by treatment with blocking solution for nonspecific binding. Subsequently the sections were stained with specific antibodies against SALL4 (1:100, Abcam) overnight. After incubation with corresponding secondary antibodies, a DAB substrate kit was used to visualize positive antigen binding. Haematoxylin counterstaining was performed and pictures were captured by microscopy.