Pan cytokeratin

MDA-MB-231, SKBR3, and MCF7 human BC cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and maintained 231 cells (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (4.5 g/L glucose) supplemented with 10% fetal bovine serum, Penicillin (100 µg/ml)/streptomycin (100 µg/mL) antibiotics. Cells were incubated under humidified conditions with 5% CO₂ at 37 °C.
Antibodies against phospho-β-catenin^S552, phospho-β-catenin^S33/S37/T41, β-catenin, E-cadherin, N-cadherin, Vimentin, CD44, phospho-GSK3β^S9, GSK3β, phospho-AKT^T308, AKT, pan-cytokeratin and Actin were purchased from Cell Signaling Technologies, Inc (Boston, MA). An anti-HNA antibody was purchased from Abcam (Cambridge, UK). p16^INK4a was purchased from Abcam.
Cycloheximide (CHX) was purchased from Sigma-Aldrich (St. Louis, MO) and NS1643 was purchased from Alomone Labs (Jerusalem, Israel).

For immunofluorescence, the paraffin-embedded endometrium tissues were sliced into 4μm sections, dewaxed with xylene and rehydrated with graded alcohol. Ethylene diamine tetra-acetic acid (EDTA) was applied for antigen retrieval. Nonspecific binding was blocked with goat serum (BOSTER, AR1009, USA). Sections were incubated with antibody to NF-L (1:200, Cell Signaling Technology, 2837T, USA) and Pan-cytokeratin (1:400, Cell Signaling Technology, 4545T, USA) at 4℃ overnight followed by secondary antibody conjugated with Alexa Fluor® 488 (1:200; Thermo Fisher Scientific). Images were obtained by fluorescence microscopy (Leica, Germany). The positive nerve fibers were counted in 10 representative fields (20x10) without knowing the histological diagnoses and clinical data. The total number of fibers of each patient were added together and further analyzed. For identification of DRG neurons, cells were fixed with 4% paraformaldehyde (PFA) and then incubated with the primary antibody MAP2 (1:200, Cell Signaling Technology, 8707T, USA) at 4℃ over-night. After that, cells were incubated with secondary antibody conjugated with Alexa Fluor® 488 (1:200; Thermo Fisher Scientific). Cells were counterstained with DAPI (Thermo Fisher Scientific) and examined under a fluorescence microscope (Leica, Germany).