Emd millipore

Cells were lysed using the mammalian protein extraction reagent, radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China), supplemented with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and PMSF (Roche Diagnostics). Proteins were separated by 10% SDS-PAGE (Sigma-Aldrich; EMD Millipore). Specific monoclonal antibodies against SATB1 (1:1,000, cat. no. ab92307, Abcam), vimentin (1:1,000, cat. no. ab92547, Abcam), E-cadherin (1:1,000, cat. no. ab11512, Abcam Inc.) and β-actin (1:5,000, cat. no. ab8227, Abcam) were applied for overnight incubation at 37°C. The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,000; cat. no. sc-2030, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). An ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell invasion was examined using a 24-well Transwell chamber (8 mm pore size; Corning, Inc.) pre-coated with Matrigel for 1-h at room temperature (EMD Millipore; Merck KGaA). Transfected HT29 and SW480 cells (50,000 cells/well) were seeded in 300 µl serum-free DMEM to the upper chamber. A total of 500 µl DMEM containing 20% FBS was added into the bottom chamber. Following incubation at 37°C for 24 h, cells on the insert were carefully removed with a cotton-tipped swab. The cells that had invaded through the membrane were stained with 0.5% crystal violet at room temperature for 10 min and photographed under an inverted microscope (magnification, ×200).

Cationic liposome was prepared as multilamellar vesicles for in vivo use as described previously (24 (link)). Briefly, 1,2 dioleoyl-3-trimethylammonium-propoane (Avanti Polar Lipids, Inc. Alabaster, AL, USA) and cholesterol (Sigma-Aldrich; EMD Millipore) were mixed in a 1:1 molar ratio, dehydrated in round-bottom tubes using a freeze dryer (SCIENTZ-50ND; Scientz Biotechnology Co., Ltd., Ningbo, China), then rehydrated in 5% glucose solution by heating at 50°C for 6 h. For the in vivo injection, plasmid DNA/lipoplexes were prepared immediately prior to injection by gently mixing cationic liposome with plasmid DNA at a ratio of 12.5 µg total cationic liposome to 2.5 µg plasmid DNA, resulting in a final concentration of 12.5 µg plasmid DNA per ml in a sterile solution of 5% glucose in water.

Total protein was extracted from BMSCs using ice-cold RIPA lysis buffer supplemented with 1 mM phenyl methane sulfonyl fluoride (Beyotime Institute of Biotechnology). Total protein was quantified using the Enhanced BCA Protein Assay kit (cat. no. P0006; Beyotime Institute of Biotechnology) and 40 µg protein/lane was separated by SDS-PAGE on 8-12% gel. The separated proteins were transferred onto a polyvinylidene fluoride membrane (EMD Millipore; Millipore Sigma) and blocked with 5% skimmed milk for 15 min at room temperature. Membranes were incubated with rabbit anti-α-SMA, anti-TGF-β1, anti-SMAD3 and GAPDH (1:1,000; cat. nos. AF0048, AF0297, AF1501 and AF1186, respectively; Beyotime Institute of Biotechnology) primary antibodies at 4˚C overnight. Following primary incubation, membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3,000; cat. no. SA00001-2, Proteintech Group, Inc.) secondary antibody at room temperature for 2 h. The bands were visualized using Millipore Immobilon ECL (Cat. No. WBKLS0100, Millipore Sigma). Protein expression was quantified using Scion Image (version 1.8.0; Scion Corporation) with GAPDH as the loading control.

The cells were harvested and a single cell suspension was prepared with H-DMEM, at a density of 1×10³-1×10⁴ cells/well. The cells were seeded into 96-well plates, with 200 µl in each well and the edge well filled with sterile phosphate-buffered saline (PBS). The cells which were divided into 5 groups and cultured at 37°C and 5% CO₂ for 24, 48 or 72 h. A total of 20 µl MTS (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) was then added to each well, and the culture was terminated subsequent to 4 h and agitated for 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm and the inhibition rate was calculated. Cell growth inhibition rate (%)=1-cell survival rate OR=(1-absorbance of the experimental group at 490 nm/absorbance of the control group at 490 nm) ×100.