Transient transfection miR-125b-inhibitor (5′-UCACAAGUUAGGGUCUCAGGGA-3′) and nonspecific control miRNA (NC, 5′-CAGUACUUUUGUGUAGUACAA-3′) were

designed based on miRbase Database (http://​www.​miRbase.​org) and synthesized by Genepharma (Shanghai, China). Cells were seeded (1.6×104/well) onto 96-well plate 18–20 h before transfection. Anti-miR-125b or NC was added to each well. After 6 h incubation at 37°C check details and 5% CO2, the medium was replaced with fresh culture medium. The cells were harvested at 48 h post transfection. Establishment of stable cell line Cells were transfected with 3 μg of plasmids (pLVTHM-MTA1-si, or pLVTHM-CTL-si) which were constructed in previous study [6], or empty pLVTHM vector using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol, then selected for the resistant to neomycin. The stable resistant cell lines were selected

and named as 95D (or SPC-A-1)/MTA1-si, 95D (or SPC-A-1)/ CTL-si, and 95D (or SPC-A-1)/NC, respectively. Quantitative real-time PCR Total RNA was extracted from the cells with Trizol reagent (Invitrogen) following the manufacturer’s instruction. Quantitative real-time PCR for miR-125b or MTA1 mRNA was performed as described previously [6]. For miR-125b quantification, U6 small nuclear RNA (U6 snRNA) was used as internal control. The primers sequences were as follows: hsa-miR-125b forward: GGCAACCTTGCGACTATAACCA,

those reverse: GTTTCCTCTCCCTGAGACCCTA; U6 snRNA forward: CTCGCTTCGGCAGCACATATACT, Selleck LY2874455 reverse ACGCTTCACGAATTTGCGTGTC. The relative quantification of expression levels was calculated using the 2−ΔΔCt method. Western blot analysis Total protein was extracted from the cells using RIPA kit (Pierce, USA). Protein concentrations of the supernatants were determined using BCA method. Equal amounts of proteins were separated by SDS-PAGE and transferred into nitrocellulose membranes, which were incubated with primary antibodies against MTA1 (1:1500; Abcam, Cambridge, MA, USA) and β-Actin (1:1000; Santa Cruz Biotech, Santa Cruz, CA, USA) at 4°C overnight. The membranes were washed three times with TBST and incubated with peroxidase conjugated goat anti-rabbit IgG secondary antibody (1:1000, Santa Cruz Biotech, Santa Cruz, CA, USA) for 1 h at room temperature. Finally, the membranes were washed three times with TBST and visualized using Western Blotting GSK461364 supplier Luminol Reagent (Santa Cruz Biotech, Santa Cruz, CA, USA) according to the manufacturer’s instruction. Wound healing assay Cells were seeded into six-well plate and grown to confluence. Wound was created by scraping confluent cell monolayers with a pipette tip. The cells were allowed to migrate for 48 h. At 0 h and 48 h after scratching, images were taken under the inverted microscope to assess the ability of the cells to migrate into the wound area.