Zf 0313

The expression of STC2 and EMT markers was observed in cultured EMT cells by an immunofluorescence microscopy. Cells grown on coverslips, which were performed in a 96-well plate, were fixed with paraformaldehyde for 30 min. After fixation, cells were washed with TBST twice and blocked in 1.5% BSA/TBST for 1 h at room temperature. Cells were respectively incubated with primary antibodies against STC2 (1:500, sc-14350, Santa Cruz), vimentin (1:500, sc373717, Santa Cruz), E-cadherin (1:500, sc-31021, Santa Cruz), N-cadherin (1:500, sc-31031, Santa Cruz) and twist (1:500, sc-81417, Santa Cruz) overnight at 4°C. Then cells were incubated with TRITC-conjugated secondary antibodies (ZF-0313, ZSGB-BIO) and FITC-conjugated ones (ZF-0314, ZSGB-BIO) for 1 h and stained with DAPI for 5 min. The images were viewed and recorded by Olympus BX40 and SPOT Flex (Diagnostic Instruments, Version 4.5).
To block ERK, PI3K signaling, 10 μM ERK inhibitor U0126 (#S1901, Beyotime) or 10 μM PI3K inhibitor LY294002 (#9901, Cell Signaling Technology) was respectively incubated with NCM460, EMT cells for 24 h to directly observe expression strength of STC2 and EMT markers. Other procedures were same as the above.

The cells were cultured on glass coverslips in 24-well plates, fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.1% Triton X-100 for 10 min. After washing with PBS, cells were blocked with nonimmune goat serum for 30 min at 25°C and then incubated with LIN41 antibody (SC-134793; 1:100 dilution; Santa Cruz Biotechnology) or anti-Flag antibodies (HT201; 1:100 dilution; Transgen BioTech) overnight at 4°C. Cells were then incubated with rhodamine (TRITC)-conjugated goat anti-mouse antibodies (ZF-0313; 1:100 dilution; ZSGB-BIO), counterstained with DAPI, and mounted.

After the imaging procedures, the solitary xenograft tumors were fixed in 4% buffered formalin. Hematoxylin and eosin (H&E) staining was performed on serial sections of 4 μm intervals for histological examination. The surface and inner samples from these samples were frozen and sectioned to detect TRAIL and EGFP expression, as previously described [28 (link)]. The sections were blocked with normal goat serum for 30 minutes (Zli-9021, ZSGB-BIO, Beijing) and then incubated simultaneously with a 1:100-diluted polyclonal rabbit anti-TRAIL primary antibody (AB2435, Abcam, U.S.A.) and a 1:20-diluted polyclonal mouse anti-EGFP primary antibody (AB16278, Abcam, U.S.A.). Our detection of TRAIL-MSCs was based on incubation with a FITC-conjugated goat anti-rabbit secondary antibody (ZF0311, ZSGB-BIO, Beijing) and a rhodamine-conjugated goat anti-mouse secondary antibody (ZF0313, ZSGB-BIO, Beijing).