E cadherin

MDA-MB-231 cells were grown on glass coverslips and were allowed to attach for 24 h prior to staining. The coverslips were washed, fixed in 3.7% (wt/vol) formaldehyde, immersed sequentially in cold methanol and cold acetone, and then allowed to air dry. The dry coverslips were incubated with diluted antibodies against E-cadherin or vimentin (Abnova), followed by incubation with a Cy3- or fluorescein isothiocyanate-conjugated secondary antibody. The nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). The coverslips were mounted with Aqua-mount (Lerner Laboratories, USA) for immunofluorescent microscopy.
The strepavidin-peroxidase (SP) method was used to detect the expression of genes of interest in clinical breast cancer samples by immunohistochemistry. Briefly, tissues were sectioned, treated with 3% H₂O₂, and then incubated in 5% goat antiserum. Primary antibodies against NFAT5 (Abcam), S100A4 (Abnova), E-cadherin (Abnova) or vimentin (Abnova) were added to serial tissue sections and incubated overnight, followed by incubation with a biotin-labeled secondary antibody. SP complex was added and then DAB-H₂O₂ was used for the color reaction before microscopy.

Protein from MCF7 and MDA-MB-231 parental or mammosphere-derived cells pre-exposed to different treatments, namely, anti-CDH11 antibody, miR-335 mimic, or miR-335 inhibitor, were loaded (20 μg/lane) and separated using 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), then the blots transferred onto polyvinylidene difluoride (PVDF) membranes, blocked in 5% skim milk in TRIS buffered saline (TBS) with Tween-20 (TBST) for 1 h, at room temperature, then incubated overnight at 4 °C with the primary antibodies against CDH11 (1:1000, clone 2C67, #H00001009-M25), E-cadherin (1:1000, clone 7H12, #MAB16359), vimentin (1:1000, clone 1G3, #H00007431-M10), b-catenin (1:2000, #MAB11143), FYN (1:1000, clone 3G11-F9, #H00002534-M01), EZH2 (1:1000, clone 2C3, #H00002146-M01), and b-actin (1:2000, clone 3G4-F9, #H00000060-M01) all purchased from Abnova (Abnova Corporation, Taipei, Taiwan). This was followed by incubation in peroxidase - conjugated secondary antibody for 1 h at room temperature, and washed with TBST three times. The signals were developed using enhanced chemiluminescence (ECL-Plus, Amersham Pharmacia Biotech, Piscataway, NJ, USA) and detected with a UVP BioSpectrum® Imaging System (Analytik Jena US LLC, Upland, CA, USA).

To determine the expression of inflammatory or airway remodeling related biomarkers in children with perennial AR, expression level of several biomarkers was measured in the serum. Blood levels of eosinophils and immunoglobulin E (IgE) were examined. Serum IL-1β (which represents the activation of inflammasomes), CCL-24 (chemokines that induce eosinophils in allergic diseases) and the EMT marker (E-cadherin, vimentin) were measured by enzyme-linked immunosorbent assay (ELISA). In addition, epithelial iNOS data were measured by a NOS kit (Cat. No. ELH-ENOS). The details of each of the ELISA kits used in the study are as follows: IL-1β (catalog No. DLB50), CCL-24 (catalog No. DCC240B), E-cadherin (ab233611), and vimentin (KA3127, Abnova). Kits were obtained from R & D Systems (Minneapolis, MN), Abcam (Cambridge, MA), and Novus Biologicals (Centennial, CO).