Anti β1 integrin

A-769662 was obtained from Abcam (Cambridge, MA), AICAR was obtained from Cell Signaling Technology (Danvers, MA). Sulfo-NHS-SS-biotin was obtained from Pierce (Thermo Fisher Scientific, Rockford, IL). Antibodies used for immunoblotting were as follows: anti-EGFR from Genetex (Irvine, CA), anti-CHC from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pACC, anti-AMPK (α1/2), anti- actin, and anti-Erk from Cell Signaling Technology (Danvers, MA), and anti-ZNF142 antibodies from Aviva Systems Biology (San Diego, CA). Antibodies used for immunofluorescence microscopy were as follows: anti-β1-integrin from EMD Millipore, Darmstadt, Germany), and anti-TfR from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunohistochemistry (IHC) was performed on 7‐, 10‐, or 20‐μm‐thick frozen skin sections fixed in 2% paraformaldehyde or ice‐cold acetone, using the following primary antibodies: anti‐pan‐keratin antibodies were as previously described (Rittié et al., 2013); anti‐Ki67 was from Biogenex (Fremont, CA, USA); anti‐β1‐integrin was from EMD Millipore (Temecula, CA, USA); and anti‐laminin‐γ2 was from AbCam (Cambridge, MA, USA). IHC was performed as previously described (Rittié et al., 2008), and slides were mounted with 90% glycerin or ProLong Gold medium (both from Thermo Fisher Scientific, Waltham, MA, USA). Imaging of horizontally cut sections was performed with a Leica MXFL III Stereo Microscope (Leica Microsystems, Buffalo Grove, IL, USA; accessed at the Microscopy and Image‐Analysis Laboratory, Biomedical Research Core Facilities, University of Michigan Medical School). Other imaging was performed with an Axioscope 2 (Carl Zeiss, Thornwood, NY, USA) equipped with a Spot digital camera (Spot Imaging Solutions, Sterling Heights, MI, USA). Large areas were captured on multiple overlapping sections, and images were merged using Photostitch 3.1 software (Canon, Lake Success, NY, USA). A microscope micrometer (Thermo Fisher Scientific) was used during imaging for image calibration.

To generate HPMCs-conditioned media, HPMCs were cultured in twelve-well plates in complete medium until they reached 80% density. Cultured media was removed, cells were washed twice, starved for 4 h in growth hormone and FBS-free medium and treated overnight in growth hormone free medium containing no FBS, FBS 10%, benign fluid 10%, malignant ascites 0.001–10%, malignant ascites 10% heat inactivated at 100 °C for 10 min, TNF-alpha (20 ng/ml) (New England Biolabs, Whitby, ON), IL-10 (1 ng/ml), IL-6 (2 ng/ml), HGF (1 ng/ml), CCL18 (20 ng/ml), CCL7, CCL8, CCL16, CCL20, CXCL1, IL1-R4 (all at 10 ng/ml) (Peprotech, Rocky Hill NJ), Leptin (0.5 ng/ml) (RnD Systems, Minneapolis MN), Actinomycin D (8 nM), NF-kB Inhibitor BAY117082 (Sigma), anti-β1 Integrin or anti-αvβ5 (5 μg/ml) (EMD Millipore, Burlington, MA). When inhibitors or Actinomycin D were used, cells were preincubated 1 h prior the treatment with the inhibitor alone and then treated overnight as described. Cells were washed three times and fresh medium without FBS nor growth factors were added in each well. HPMCs were cultured for 24–96 h and medium conditioned by stimulated HPMCs were subjected to CA125 quantification. Results were normalized to the total protein concentration in the cell lysate and expressed as kilounits of MUC16 per gram of total proteins.