Ab39184

Protein was extracted from fresh NIP tissue using ice-cold RIPA lysis buffer (Abcam) mixture containing protease and phosphatase inhibitor (Sigma Aldrich). After 30 minutes of incubation on ice with shaking, followed by centrifugation at 14,000 rpm, the supernatant containing the protein was aspirated for use in Western blot analysis. Protein concentration was first determined using BCA protein assay (Thermo Fisher, USA); then, 4X laemmli sample buffer was added to the protein samples according to the manufacturer’s instructions, and heated at 95°C for 10 minutes. 30 μg total protein from each sample was subjected to electrophoresis in 10% SDS-PAGE gel and then transferred onto a 0.22μm PVDF membrane (Millipore, USA). After 2 hours non-specific blocking with 5% skimmed milk, primary antibody of MMP-1 (1:2000, Abcam, ab137332), MMP-7 (1:2000, Abcam, ab207229), MMP-9 (1:4000, Abcam, ab76003), TIMP-1 (1:2000, Abcam. Ab109125), TIMP-3 (1:2000, Abcam, ab39184), GAPDH (1:5000, Abcam, ab186930) were added to the membrane and incubated at 4°C overnight. HRP-conjugated anti-rabbit IgG or anti-mouse IgG were then added as secondary antibody at room temperature for 1 hour. After secondary antibody staining, ECL reagent (Bio-Rad, USA) was used to visualize the blots. Densitometry was performed with ImageJ software to quantify the protein amounts of each sample.

DADS, purchased from Fluka Co. (Milwaukee, Wisconsin, USA), was dissolved in Tween-80 and stored at −20 °C after a 100-fold dilution with saline. The primary antibodies against RORα (ab60134), MMP-9 (ab38898), TIMP3 (ab39184), vimentin (ab92547), E-cadherin (ab40772), snail (ab53519), Ki-67 (ab66155), and CD34 (ab81289) were provided by Abcam (Cambridge, MA, UK). The primary anti-bodies against Wnt-1 (sc-514531), β-catenin (sc-1496), Axin (sc-14029), c-Jun (sc-44), c-Myc (sc-40), TCF-4 (sc-271287), β-actin (sc-8432) and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibody against p-β-catenin was purchased from Cell signaling technology (Danvers, MA, USA). pGL3-c-Myc promoter luciferase reporter plasmid was obtained from Guangzhou Cyagen Biosciences Inc.

Samples, including cell culture samples and cryosectioned tissues, were rinsed with 1 × PBS (Gibco) and treated with 0.3% Triton X-100 (Sigma-Aldrich) to increase permeability. After incubation in blocking buffer comprised of 1 × PBS (Gibco), 1% BSA (Sigma-Aldrich), and 0.3% Triton X-100 (Sigma-Aldrich) for 1 hour, samples were incubated with the diluted primary antibodies (1:100) followed by the corresponding secondary antibodies (1:200). Cell nuclei were stained with DAPI (Santa Cruz). Samples were mounted using an anti-fluorescence quenching agent (Solarbio, Beijing, China) and imaged by confocal microscopy (Nikon).
The primary antibodies include rabbit polyclonal anti-RFP (Abcam, ab185921), rabbit polyclonal anti-Cx43 (Abcam, ab11370), rabbit polyclonal anti-TIMP3 (Abcam, ab39184), rabbit polyclonal anti-SOX2 (Abcam, ab97959), rabbit monoclonal anti-Nanog (Abcam, ab109250), and rabbit polyclonal anti-LAMA4 (Abcam, ab209675). The secondary antibody was Alexa Fluor 568 goat anti-rabbit immunoglobulin G (IgG; Invitrogen).