Vimentin

17β-Estradiol (E₂), cobalt chloride (CoCl₂) (Sigma Aldrich, St. Louis, MO, USA)). Mocetinostat, GSK-126, and GSK-J4 inhibitors were as in the literature [49 (link),55 (link),56 (link)]. Antibody to β4 integrin (450-11A) was as in the work of [71 (link)]. Antibodies: E-cadherin (GeneTex, Irvine, CA, USA; GTX100443), ERβ (L-20, Santa Cruz Biotechnology, Dallas, TX, USA; SC-6822, CWK-F12, DSHB), eNOS (BD Biosciences, Franklin Lakes, NJ, USA; BD610297), EZH2 (D2C9, Cell Signaling, Danvers, MA, USA; #5246s), H3K27me3 (Active Motif, Carlsbad, CA, USA; #39156 and #39155), HIF-2α (Novus Biologicals, Centennial, CO, USA; NB100-132), HIF-1α (Novus Biological NB100-105), N-cadherin (Thermo-Fisher, Waltham, MA, USA; #PA5-19486), integrin β3 (Genetex, Irvine, CA, USA; GTX111672), Snail (Genetex GTX125918), Vimentin (Bethyl, Montgomery, TX, USA; A301-620A-T), Laminin beta 1 (Abcam, Cambridge, UK; ab108536), Fibrillarin (Thermo Fisher MA3-16771), HSP90 (StressMarq, Victoria, BC, Canada; SPC-104), IgG (Normal Rabbit, Millipore, Burlington, MA, USA #12-370, Normal Rabbit, Bethyl #S120-101), Tubulin (DM1A, Abcam #ab7291), β Actin (Abcam).

Total protein extracts were obtained using RIPA buffer and nuclear/cytosolic extracts were prepared as in the work of [39 (link)]. Western blot assay was performed using 40 μg of total extract and proteins were resolved by SDS-PAGE using 7.5% or 4%−12% gradient Invitrogen Precast gel (NuPage and MES buffer). The following primary antibodies were used: E-cadherin (1:2000, GeneTex GTX100443), β4 integrin 450-11A (1:1000), HIF-2α (1:500, Novus Biologicals, Centennial, CO, USA; NB100-132), HIF-1α(1:500, Novus Biological NB100-105), N-cadherin (1:5000, Thermo-Fisher PA5-19486), integrin β3 (1:1000, Genetex GTX111672), Snail (1:1000, Genetex GTX125918), Vimentin (1:1000, Bethyl A301-620A-T), Fibrillarin (1:1000, Thermo Fisher MA3-16771), Laminin beta 1 (1:1000, Abcam ab108536), Tubulin (1:5000, DM1A Abcam #ab7291), β Actin (1.1000, Abcam), and HSP90 (1:1000, StressMarq SPC-104C). Specific protein signals were revealed with ECL Prime (Amersham, GE Healthcare, Boston, MA, USA) and detected by UVIDOC (Eppendorf S.r.l., Hamburg, Germany). The intensity of each band was evaluated using the NIH Image J 1.8 software (National Institutes of Health, Bethesda, MD, USA).