Anti slug

For the immunocytochemistry assay, SiHa-Vec and SiHa-Slug cells were seeded onto cover slips for 48 h, washed with PBS three times, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 20 min at room temperature. The cover slips were incubated with anti-EpCAM (1:200 dilution, sc-25,308, Santa Cruz, USA) and anti-Slug (1:50 dilution, #9585, Cell Signaling Technology, USA) at 4 °C overnight, and a horseradish peroxidase-conjugated secondary antibody was added for 30 min at room temperature.
For the immunofluorescence assay, after incubation with anti-EpCAM (1:50 dilution, sc-25,308, Santa Cruz, USA) and anti-Slug (1:50 dilution, #9585, Cell Signaling Technology, USA) overnight at 4 °C and washing with PBS, the cover slips were incubated with fluorescently conjugated secondary antibodies (Alexa Fluor 555, #A-31,570 and Alexa Fluor 488, # A-21,206, Thermo Fisher Scientific, USA) for approximately 60 min at room temperature. Finally, the cells were washed with PBS and incubated with DAPI (Solarbio). Cell images were taken with a laser scanning confocal microscope (Leica). The cells were imaged with an Olympus CX31 microscope digital camera and Leica DFC 500 digital camera and processed with ImageJ software.

Cells were lysed in buffer containing 150 mM NaCl, 1.0% nonidet-P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0, and a protease inhibitor cocktail (Roche Applied Science, Vienna, Austria, pH 7.4). Frozen tissue samples stored in liquid nitrogen were cut into pieces with scissors. Each sample was homogenized in lysis buffer at a ratio of 1:20 w/v. After a centrifugation at 13,000 rpm for 20 min step the supernatant was used to measure the total protein. Electrophoresis was performed as described previously^{20 (link)}. The following primary antibodies were used for western blot analysis: anti-LAMB3 (1:1000; OriGene Technologies Inc. Rockville, MD, USA) anti-phospho-Akt (Ser 473), anti-total Akt, anti-vimentin, anti-Slug, anti-Snail, anti-β-actin (1:1,000; Cell Signaling Technology Inc, Danvers, MA, USA), LAMA3, LAMC2, anti-E-cadherin, and anti-N-cadherin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000; Santa Cruz Biotechnology), immunoreactive bands were visualized by enhanced chemilumi-nescence (ECL) detection.

Anti-vimentin, anti-Snail, and anti-Slug antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NUCB2, anti-Twist1, and anti-Ki-67 antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-E-cadherin, anti-p21^waf1, anti-BCL2, and anti-cyclin D1 antibodies were from Dako (Copenhagen, Denmark). Anti-p27^kip1, anti-Rb, anti-X-linked inhibitor of apoptosis (XIAP), anti-BAX, and anti-N-cadherin antibodies were from BD Biosciences (San Jose, CA, USA). Anti-ZEB1 and anti-β-actin antibodies and adriamycin (ADR: D1515) were from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-phospho (p) Rb at Ser807/811, anti-cleaved caspase-3, and anti-poly (ADP-ribose) polymerase 1 (PARP1) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cyclin A2 and anti-cyclin B1 antibodies were from Novocastra (Newcastle, UK) and Santa Cruz Biotech (Santa Cruz, CA, USA), respectively.