Mouse anti snail

Cells were lysed and subjected to western blot analysis as described by us [51 (link)]. Antibodies were as follows: mouse anti-TIF1γ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-E-cadherin, anti-N-cadherin and anti-Vimentin (BD Biosciences, San Jose, CA, USA), mouse anti-Snail (Cell Signaling Technology, Danvers, MA, USA), mouse anti-β-actin and anti-mouse secondary antibodies (Santa Cruz Biotechnology). Molecular sizes of TIF1γ, E-cadherin, N-cadherin, Vimentin, Snail and β-actin proteins shown on the immunoblots are 150kD, 120kD, 130kD, 57kD, 29kD and 43kD, respectively. Each experiment was carried out in triplicate.

The CSC were collected at different time points after transfection, and total proteins were extracted using a total protein extraction kit (ProMab). After quantification of protein concentrations using a BCA assay (Santa Cruz Biotech), the individual cell lysates (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free dry-milk in TBST and incubated with rabbit anti-Slug (1:500; Cell Signaling Technology), mouse anti-vimentin (1:400), mouse anti-GAPDH (1:800), goat anti-E-cadherin (1:500; Santa Cruz Biotech), mouse anti-Snail (1:500; Cell Signaling Technology), rabbit anti-N-cadherin (1:500), and rabbit anti-CK 18 (1:5000; Abcam) at 4°C overnight, respectively. After washing, the bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG at room temperature for 1 h and visualized using enhanced chemiluminescence. The relative levels of targeting proteins to the control GAPDH were determined using ImmuNe software.

Cells were hydrolyzed in RIPA buffer with a mixture of protease inhibitors and phosphatase inhibitors. Protein products were isolated by SDS–PAGE and transferred onto nitrocellulose membranes. We cut the membranes according to the molecular weight of the target proteins. The membranes were then blocked in TBST buffer with 3.0% BSA for 1 hour and incubated at 4 °C with primary antibodies overnight. The membranes were rinsed three times with TBST buffer and incubated at room temperature for 2 hours with the corresponding secondary antibody. Protein detection was performed using an enhanced chemiluminescence system. The expression of proteins of interest was normalized to that of β-actin. The following primary antibodies used in WB analysis: anti-mouse snail, anti-mouse Vimentin, anti-mouse E-cadherin, and anti-mouse N-cadherin (Cell Signaling Technology, Danvers, MA, USA); anti-rabbit CST1, anti-mouse GAPDH, and anti-mouse β-actin (ABclonal, Biotechnology Co. Ltd. Wuhan, China); and anti-rabbit or anti-mouse secondary antibodies (Beyotime Biotechnology Co. Ltd. Shanghai, China). Relative protein expression was normalized to that of β-actin. The same set of lysate samples were run in parallel (sister) gels to test different proteins (same for all figures).

For immunofluorescence, we fixed the cells with 4% paraformaldehyde for 15 min at room temperature, followed by blocking for nonspecific binding antigen using 1% PBS/BSA, and then incubated overnight with anti-mouse Snail (Cell Signaling Technology clone L70G2, CST: Danvers, MA, USA) or anti-rabbit H3K9 (Cell Signaling Technology clone C5B11) antibodies. Subsequently, cells were washed and incubated with anti-mouse Alexa-Fluor 488 or anti-rabbit Alexa-Fluor 647 conjugated secondary antibodies (Thermo Fisher, Waltham, MA, USA). DNA content was stained with Hoechst 33,342 (Invitrogen) for nuclear visualization and propidium iodide (PI—Invitrogen, Paisley, UK) to assess cell death. Images were captured using the ImageXpress Micro 4 system.