Nfat5

MDA-MB-231 cells were crosslinked with formaldehyde and harvested for chromatin immunoprecipitation (ChIP) as described [18 (link)]. Briefly, chromatin was fragmented by sonication, and pre-cleared chromatin was immunoprecipitated overnight with monoclonal antibodies against NFAT5 (Abcam, UK), EZH2 (Millipore, Darmstadt, Germany), LSD1 (Abcam), trimethylated histone H3K27 (Millipore), dimethylated histone H3K4 (Abnova) or corresponding IgG isotype control (Abcam). The enrichment of specific DNA fragments was analyzed by PCR with primers flanking the S100A4, VEGF-C or miR-568 promoter region.

Briefly, cells at three days were lysed in the RIPA buffer to collect the total proteins. Then samples were separated using SDS-PAGE electrophoretic gels (10%). After electrotransferring onto PVDF membranes, the blots were treated with different primary antibodies [NFAT5 (Abcam, Cambridge, MA), PAI-1 (Abcam, Cambridge, MA), PLAT (Proteintech, Chicago, IL), PLAU (Proteintech, Chicago, IL), PLG (Proteintech, Chicago, IL) or β-actin (Abcam, Cambridge, MA)] and horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO, Beijing, China). Protein bands were finally treated with ECL substrate solution and exposed to blue X-ray film to obtain the images.

Cells were harvested at the desired times, and proteins were extracted, separated on an SDS/PAGE gel, transferred onto polyvinylidene fluoride membranes, and subjected to immunoblot analyses. Blotting was performed using antibodies targeting NFAT5 (Abcam, Cambridge, UK), S100A4, E-cadherin, vimentin (all from Abnova, Taiwan, China), α-catenin (Santa Cruz, Dallas, TX, USA), β-catenin, Akt, phosphorylated Akt, ERK, phosphorylated ERK (all from Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA).

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.

Immunofluorescence staining was carried out using antibody against NKCC1 (Santa Cruz Biotechnology, CA, United States; used at 1:50), NFAT5 (Abcam, Cambridge, MA, United States; used at 1:100) and HIF-1α (Abcam, Cambridge, MA, United States; used at 1:100) in peri-ischemic hippocampal brain slices and in primary cultured hippocampal neurons. Microtubule-associated protein 2 (MAP2, ABclonal, Wuhan, China; used at 1:200) was used as the neuronal mark. The protocol is detailed in the Supplementary Material.