Ab29112

For immunofluorescence analysis, tissues were stained with CD8 antibody (ab60076, abcam, Cambridge, USA), PD1 antibody (ab137132, abcam), SALL4 antibody (ab29112, abcam), hepatocyte specific antigen antibody (GTX73779, Genetex), CD45 antibody (MA5-17687, Thermo Scientific, Waltham,USA) and PD-L1 (ab205921, abcam) followed by Alexa Fluor 647 conjugated goat anti-rat IgG (A-21247), Alexa Fluor Plus 488 conjugated goat anti-rabbit IgG (A-32731) and Alexa Fluor Plus 555 conjugated donkey anti-mouse IgG (A-32727) (Thermo Scientific). Images were acquired on a Zeiss 710 Meta multi-photon confocal microscope (Zeiss, Oberkochen, Germany). To assess the immunostaining quantification, we analyzed the slides via an image analysis workstation (Image Pro Plus 6.0, Media Cybernetics). Mouse liver tissues were collected and embedded in OCT. Intrahepatic HBsAg, Pd-l1, or Sall4 expression was visualized by immunohistochemical staining with rabbit anti-mouse HBs Ab (Genetech, Shanghai, China) or rabbit anti-mouse Pd-l1 Ab (eBioscience, San Diego, CA, USA), or anti-Sall4 Ab (abcam, cat#57577) followed by Envision System HRP detection staining (Genetech, Shanghai, China) performed according to the manufacturer’s protocol. Liver sections were stained with hematoxylin. Images were taken with an OLYMPUS microscope.

A RIPA buffer containing 1% protease inhibitors was used to lyse the cells. SDS-PAGE was used to separate the protein sample, followed by the transfer to PVDF membranes. The membrane was blocked with 5% bovine serum albumin and then incubated overnight with specific antibodies against SALL4 (ab29112, Abcam), VEGF-A (66828-1-Ig, Proteintech), VEGF-B (YT4871, Immunoway), VEGF-C (22601-1-AP, Proteintech), and GAPDH (MB001; Bioworld Technology, St. Louis Park, MN, USA). After 2 h of incubation with the secondary antibodies (Bioworld Technology) at room temperature, the bands were visualized with a chemiluminescent detection system.

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.

The chromatin immunoprecipitation (ChIP) assay was performed in MGC-803 and SGC-7901 cells by using a commercial kit (Millipore, Darmstadt, Germany). After cross-linking with 1% formaldehyde at 37 °C for 10 min, the cells were harvested in sodium dodecyl sulfate lysis buffer and the DNA was shredded to fragments of 200 bp by sonication. The pre-cleared chromatin was incubated with 1 µg anti-SALL4 (ab29112, Abcam) or non-specific IgG overnight. Protein G-agarose beads were added and incubated at 4 °C for 1 h. After reversing the cross-links, the DNA was isolated and used for PCR. The information of the sequences of ChIP primers are listed in Additional file 2.

The cells were washed twice with PBS and lysed with RIPA buffer containing 1% protease inhibitors. Equal amounts of proteins were separated on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes, followed by blocking with 5% nonfat milk for 1 h. The membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-SALL4 (1:500, ab29112, Abcam), anti-HK-2 (1:1000, 2867T, Cell Signaling Technology), anti-LDHA (1:1000, 3582T, Cell Signaling Technology), anti-PKM2 (1:1000, 4053T, Cell Signaling Technology), anti-β-actin (1:1000, 4970T, Cell Signaling Technology). After incubation with the secondary antibodies (Bioworld Technology) at 37 °C for 1 h, the bands were visualized with a chemiluminescent detection system.