Anti vimentin

Proteins were extracted from frozen tissues according to standard protocols. To collect secreted proteins, Hsp90α+/+ and hsp90α−/− primary cell lines or MDA-MB-231 cells were seeded at the same density in OPTI-MEM medium (ThermoFisher Scientific) with 2% FBS; after 24 hours, cells were washed with PBS and grown for 48 hours in OPTI-MEM medium without FBS. Media were collected and centrifuged at 12 000 rpm for 20 minutes at 4°C, supernatant was concentrated using centrifugal filter units (10 kDa cutoff, Amicon Ultra) according to manufacturer's instructions.
Equal amounts of protein extracts or 40 μl of conditioned medium were subjected to SDS-PAGE followed by standard immunoblotting procedures. The following primary antibodies were used: anti-Hsp90α (EMD-17D7, Merck Millipore), anti-Trap1/Hsp75 (BD Biosciences), anti-Hsp90β (H9010, ThermoFisher Scientific), anti-Vimentin (PA5-27231; ThermoFisher Scientific), anti-E-cadherin (H-108, Santa Cruz Biotechnology), anti-PyMT antibody (sc-53481; Santa Cruz Biotechnology) and the pan-actin antibody C4 (Millipore). The secondary antibodies used were horseradish peroxidase-goat anti-rabbit or anti-mouse immunoglobulins (Dako).

For immunohistochemical staining, the slides were incubated overnight with anti-acetyl histone H3 (Cell Signaling, Danvers, MA, USA) and then anti-rabbit secondary antibody for 60 minutes at RT (Vector laboratories, Burlingame, CA, USA). HRP was detected using the Vector DAB detection system, and the slides were counterstained with Mayer's hematoxylin. For immunofluorescence assays, the samples were fixed with methanol at −20°C for 6 minutes, blocked with 0.5% (v/v) Triton X-100 in PBS and 3% (w/v) bovine serum albumin (BSA) and incubated with anti-Vimentin (Thermo Scientific, Waltham, MA, USA), anti-BMI-1 (Millipore, Billerica, MA, USA), anti-Pan-cytokeratin (Cell Signaling, Danvers, MA, USA), anti-phospho S6 (Cell Signaling, Danvers, MA, USA) and ac.H3 (Lys9) (Cell Signaling, Danvers, MA, USA). Cells were then incubated with FITC- or TRITC-conjugated secondary antibody and stained with Hoechst 33342 (Sigma-Aldrich Corp., St. Louis, MO, USA) to visualize DNA content. Images were taken using a QImaging ExiAcqua monochrome digital camera attached to a Nikon Eclipse 80i Microscope (Nikon, Melville, NY) and visualized with QCapturePro software.

Total cell lysates were obtained by incubating cells in a lysis buffer containing 1% v/v Triton, 0.1% w/v SDS, 2 mM CaCl₂, and 100 μg/ml phenylmethyl-sulfonyl-fluoride. Protein content was determined using the Protein Assay Kit 2 (Bio-Rad Laboratory, Hercules, CA, USA). Sixty micrograms of proteins were electrophoresed in 10% SDS–polyacrylamide gel and then electrotransferred to nitrocellulose membrane (Whatmann, Dassel, Germany). The membrane was incubated with 1 μg/ml primary antibody and then with appropriate horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a chemiluminescent detection system (Thermo Scientific, Rockford, IL, USA) and signals were digitally acquired by Chemidoc XRS system (BIORAD). Antibodies anti-β-actin, MCT1, MCT4, COX1, β-tubulin were from Santa Cruz Biotechnology, anti HIF-1α were from Becton Dickinson (Franklin Lakes, NJ, USA), anti-AMPKalpha and p-AMPKalpha (Thr172) were from Cell Signaling Technology, Inc. (Danvers, MA, USA), anti-vimentin were from Thermo scientific (Waltham, MA, USA), anti-αSMA were from Sigma. Densitometric analysis of protein bands was performed using the ImageJ software. Relative values were calculated by comparison with experimental control, defined as 1, and normalized by the corresponding values of loading control (actin or β-tubulin).