HEC-1A, Human endometrial adenocarcinoma cells (HTB112, ATCC, Manassas, VA) were cultured in McCoys media, supplemented with 10% fetal calf serum, 1% L-Glutamine, 1% penicillin/streptomycin at 37°C in a humidified 5% CO2 incubator. Cells were passaged every 3-4 days. HEC-1A cells were stably transfected with expression plasmids for a control eGFP, or a human PMP22 open reading frame tagged with a myc-epitope in the second extracellular loop using FuGENE 6 (Roche Molecular Biochemicals) per the manufacturer's instructions. These expression vectors have been previously described [17, 25]. Stable cellular clones were selected using Geneticin (800 ug/ml; Invitrogen, Carlsbad, CA) and referred to as HEC-1A/Vector (empty vector control), and HEC-1A/PMP22 (PMP22 overexpression). In some experiments, PMP22 levels were decreased by transiently transfecting HEC-1A cells with 75 picomoles PMP22 siRNA duplexes (SMARTpool L-010616-00-0005; ThermoScientific, Boulder, CO) and a lipophilic transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, California) for 48 hours. As a negative control, cells were transfected with 75 picomoles scrambled control siRNA (D-001206-13-05; ThermoScientific). Cells were harvested for Western blot, flow cytometry, immunofluorescence or immunoprecipitations 36-48 hours post-transfection, as detailed below.
Retrospective human tissue was obtained from the UCLA Department of Pathology and Laboratory Medicine Tissue Procurement Core Facility (TPCL) under an exemption from the Institutional Review Board. To validate PMP22 expression in human endometrium, we tested total PMP22 expression in whole tissue fragments of proliferative endometrium (n = 6) and secretory endometrium (n = 6). As controls, we also obtained normal frozen tonsil and lung. Tissue samples were collected and frozen in the -80 C. These tissues were prepared in the hospital biorepository, and clinical annotation is available though a database. Cases were classified and selected based on a normal diagnosis using the Anatomic Pathology system, and no information regulated by HIPPA was included in the study, which qualifies for the status of NIH Exemption #4.
All tissue was homogenized and RNA isolated using the Qiagen RNAeasy kit per manufacturer's instructions. The Qiagen OneStep RT-PCR kit (Valencia, CA) was employed using human PMP22 exon 1A or 1B  or GAPDH -specific primers using 1 μg RNA.
PMP22 1A: 5'- TTACAGGGAGCACCACCA-3'
PMP22 R: 5'-ATCATGGTGGCCTGGACA-3'
The RT-PCR conditions were reverse transcribed at 50°C for 30 min followed by PCR (25-40 cycles) at 95°C (30 s), 55°C (30 s), and 72°C (1 min).
In order to quantititate mRNA levels, the Qiagen QuantiTect SYBR Green RT-PCR Kit was utilized. Primers were synthesized (Real Time Primers, Elkins Park, PA) which could recognize both transcripts of PMP22. The 25 μl PCR reaction included 12.5 μl 2 × RT-PCR Master Mix, 0.5 μmol/L forward primer, and 0.5 μmol/L reverse primer. The reactions were incubated in a 96-well plate and reverse transcribed and amplied on the Applied Bio Systems7500 Fast System (Carlsbad, CA) using the following primers:
PMP22 F: 5'- GTATCATCGTCCACGTC-3'
PMP22 R: 5'-GGCAGAAGAACAGGAACAGA-3'
The 2-⊿⊿Ct method for relative quantification of gene expression was used to determine PMP22 expression levels . Each sample was analyzed in triplicate, and the housekeeping gene GAPDH was used to normalize expression as previously described . Differences between PMP22 expression in proliferative and secretory endometrium was determined using Mann-Whitney U test.
Western blot analysis
Tissue or cell lines were lysed in Laemmli buffer as previously described  and analyzed for PMP22 expression by SDS-PAGE Western blot analysis. Approximately 25 μg of protein were loaded per lane. In order to detect PMP22 expression, samples were treated for 2 h at 37°C with PNGase (New England Biolabs, Beverly, MA) to remove N-linked glycans . Proteins were then separated by SDS-PAGE and transferred to a nitrocellulose membrane (Invitrogen). Following transfer, membranes were stained with Ponceau S (Sigma-Alrich, St. Louis, MO) to validate the efficiency of transfer. A solution of 10% nonfat milk in Tris buffered saline containing 0.1% Tween-20 was used to block non-specific binding. Membranes were probed with rabbit anti-PMP22 antisera  or with anti-β-actin antibodies (Sigma-Aldrich). Protein bands were visualized using a horseradish peroxidase-labeled secondary antibody (Southern Biotechnology Associates, Birmingham, AL) followed by chemiluminescence (ECL; Amersham Biosciences). Band intensities were quantified using the NIH program Image J. Blots were digitized using a flatbed scanner and the band density measured using Image J. The relative intensity of PMP22 in the three cell lines was calculated by dividing the volume intensities over that of the β-actin control. Experiments were repeated three times and averaged.
Cells were washed in PBS and solubilized in lysis buffer for 30 min at 4°C. (1% Nonidet P-40 containing 2 mM phenylmethylsulfonyl fluoride, 10 ug/ml aprotinin, 2 ug/ml pepstatin, 10 mM iodoacetamide, 0.1 mM EDTA, 10 mM HEPES, and 10 mM KCl). Lysates were sonicated for 15 seconds and pre-cleared by incubation with protein A-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Pre-cleared lysates were incubated overnight with Protein-A beads bound to either anti-PMP22 rabbit polyclonal antisera (Sigma-Aldrich), anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology), or control rabbit sera. The beads were washed once in lysis buffer and 50 mM Tris buffer to neutralize salt. Immune complexes were eluted from the beads with Laemmli buffer. Cell lysates were subsequently analyzed by Western blots.
HEC-1A cells were plated onto glass coverslips (Fisher Scientific, Pittsburgh, PA) and incubated for 24 h at 37°C. Cells were fixed in cold methanol, blocked in 1% normal goat serum and washed in PBS with 0.01% Triton X-100. Cells were subsequently incubated at 4°C with PMP22 rabbit antisera (1:100; Sigma, CA), anti-α6 integrin rat antibody (1:100; BD Biosciences) in a humidified chamber. Cells were rinsed with PBS with 0.01% Triton X-100, then incubated for 1 hour at RT with Alexa Fluro 488-conjugated goat anti-rabbit IgG (1:50), or fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG2a (1:450; eBiosciences). Negative controls included incubation with secondary antibody alone. Cells were briefly washed in PBS with 0.01% Triton X-100, double deionized H2O, and mounted onto slides in a 3.5% n-propyl gallate-glycerol solution. Mounted coverslips were analyzed for PMP22 and α6 integrin localization using confocal microscopy.
The membrane expression of α6 integrin, α2 integrin, and αvβ3 integrin were assessed by flow cytometry. Cells were fixed in 2% paraformaldehyde (w/v) in PBS for 20 min on ice. Cells were pelleted and resuspended in PBS with 2% fetal calf serum (FCS). Cells were incubated with primary antibody (1:100) and incubated at RT for 20 min. Cells were then incubated with red-phycoerythrin-conjugated anti-rat Ig-κ light chain antibody or red-phycoerythrin-conjugated anti-mouse Ig-κ light chain antibody for 20 min (0.25 ug/ 106 cells; BD Biosciences). Negative control cells were treated similarly, but without primary antibody. After two consecutive washes with PBS, cells were resuspended in PBS and analyzed with a FACScan flow cytometer (BD Biosciences). Integrin expression levels were calculated as mean fluorescent intensity (MFI) using CellQuest software. Experiments were performed in triplicate.
Retrospective formalin fixed, paraffin embedded normal human proliferative or secretory endometrial samples were obtained from the TPCL at UCLA. Whole tissue sections of proliferative endometrium (n = 6) or secretory endometrium (n = 6) were analyzed by immunohistochemistry for PMP22 or α6 integrin expression. Briefly, paraffin-embedded human tissue samples were deparaffinized, blocked for endogenous peroxidase activity with 3% hydrogen peroxide, and heated at 95°C for 20 min with citrate buffer (19). The tissue samples were blocked with normal goat serum in TBS with Tween for 10 min and stained with rabbit-anti-PMP22 polyclonal antibody (1:100; Sigma-Aldrich) or anti-α6 integrin rabbit antibody (1:100; Santa Cruz Biotechnology Inc., Santa Cruz CA) overnight in a humidified chamber. Slides were developed by incubation with a biotinylated secondary antibody from the Vectastain Elite ABC kit (Vector Laboratories) according to the manufacturer's protocol, followed by a diaminobenzidine tetrahydrochloride (DAB) substrate solution (Pierce, Rockford, IL). Nuclei were counterstained with hematoxylin. Slides were analyzed for PMP22 and α6 integrin expression by microscopy.
To detail the staining of PMP22 and α6 integrin in proliferative and secretory endometrium, a semi-quantitative analysis was performed. Each section stained was assessed by considering the staining intensity (0 = below the level of detection, 1, weak; 2, moderate; and 3, strong) and the percentage of cells staining at each intensity level (0-100%). For each tissue, an integrated value of intensity combined with frequency was derived using the formula: [(3x) + (2y) + (1z)] / 100 where x, y, and z are % staining at intensity 3, 2, and 1, respectively. Differences in expression between proliferative and secretory endometrium were determined using a Mann-Whitney test, where p < 0.05 was considered significant.
A standard static cell adhesion assay (15-20 min) was performed as previously described . Briefly, 96-well plates were precoated for 2 hours at 37°C with the ECM substrates laminin, fibronectin, poly-D-lysine (positive control; Roche Molecular Biochemicals; 5-10 μg/ml), or 1% fatty acid-free bovine serum albumin (negative control; Sigma-Aldrich). Cells (7 × 104) were plated onto the ECM in serum-free conditions and incubated at 37°C for 30 min. Unbound cells were washed away. Bound cells were fixed, stained with toluidine blue and then lysed using 2% SDS (Biowhittaker, Walkersville, MD). The resultant soluble toluidine blue was quantitated by measuring the absorbance at 595 nm. Binding to each ECM was performed in triplicate. Each experiment was repeated at least three times. An unpaired Student's t test was used to confirm significance between HEC-1A/PMP22, HEC-1A/Vector, HEC-1A/scrambled siRNA control, and HEC-1A/PMP22 siRNA cells.
In antibody blocking experiments, cells were preincubated with various dilutions of anti-α6 integrin  or anti-α2 integrin  function blocking antibodies (BD Biosciences) for 60 min at 4°C. Cells (5-7 × 104) were plated in triplicate into a 96-well plate precoated with laminin or poly-D-lysine and allowed to adhere for 30 min. Unbound cells were washed away, and bound cells were quantitated as described above. Each experiment was repeated three times.