Meg 3

PANC-1 and Aspc-1 cells were treated with pWPXL-miR-148a or pWPXL-control for 24 h at 37°C, homogenized in lysate buffer containing protease-inhibitor and centrifuged at 6,000 × g at 4°C for 10 min. The supernatant of was used for analysis of the total protein concentration using bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). For detection, proteins (40 µg) were loaded and separated using 12% SDS-PAGE gels, transferred to nitrocellulose membranes and hybridized as previously described (30 (link)). Subsequent to blocking in 5% skimmed milk, membranes were probed with primary antibodies MEG-3 (1:500; 5122 Cell Signaling Technology, Inc.), E-cadherin (1:1,000; ab11512), Vimentin (1:1,000; ab92547), Snail2 (1:1,000; ab180714), β-catenin (1:1,000; ab32572), C-myc (1:1,000; ab32072), Cyclin D1 (1:1,000; ab134175) and β-actin (1:1,000; ab8226) and incubated for 1 h at 37°C, followed by incubation with HRP-conjugated goat anti-mouse secondary antibody (1:2,000; ab6785; all Abcam) for 24 h at 4°C. The blots were visualized using a chemiluminescence kit (Thermo Fisher Scientific, Inc.). Quantity of protein was analyzed using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.).

PANC-1 and Aspc-1 cells grown on lysine-coated glass coverslips were treated with pWPXL-miR-148a or pWPXL-control for 48 h. Subsequently, the cells were fixed with 4% paraformaldehyde, followed by blocking in 1% bovine serum albumin and 0.1% Triton X-100 in PBS for 60 min at 37°C. The pancreatic tumor cells were then incubated with antibodies against MEG-3 (1:500 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h at 25°C in a humidified atmosphere. Subsequently, the cells were incubated with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:400 dilution; cat. no. 4412; Cell Signaling Technology, Inc.) after washing with PBS. Pancreatic tumor cell nuclei were stained with DAPI (10 mg/ml) for 30 min at 25°C in a humidified atmosphere. The PANC-1 and Aspc-1 cells were mounted in anti-fade mounting medium and images were captured using a Zeiss Confocal Spectral microscope (magnification, ×40; Carl Zeiss, Jena, Germany).