Burwell et al

							                         Burwell et al. Supplemental Material

MATERIALS AND METHODS



Cell Culture, Plasmids, and Transfection.

H16N-2 mammary epithelial cells were obtained from Dr. Saraswati Sukumar (Johns

Hopkins University) and grown in MEGM (Cambrex/Clonetics, East Rutherford, NJ),

which is MEBM medium supplemented with human epidermal growth factor, bovine

pituitary extract, and insulin. The MEGM is supplemented with 10% fetal bovine serum

(Invitrogen, Carlsbad, CA). Cells were transfected with plasmids that contain the cDNA

for WT1 (-Ex5/-KTS) or WT1 (+Ex5/+KTS) under the control of the CMV immediate

early promoter (designated pCB6WT(-/-) and pCB6WT(+/+) respectively) or under the

control of the sheep metallothionein promoter (designated pMTWT(-/-) and

pMTWT(+/+) respectively). Control plasmids containing the same promoters with no

cDNA insert (pcDNA3.1 and pMTCB6, respectively) were also used. Tissue culture

plates were seeded with 106 cells, and cells were transfected with 15 µg of DNA with

TransIT LT1 (Mirus, Madison, WI). Successfully transfected cells were selected in 1

mg/ml Geneticin (Invitrogen), and individual colonies were isolated and propagated.



Immunofluorescence Microscopy.

Stably transfected H16N-2 cells were seeded into 4-chambered glass slides (Nalge, Lab-

Tek II Chamber Slide System, Nalge Nunc International, Rochester, NY) at 5 x 104 cells

per chamber. Cells were then incubated overnight with 75 µM ZnCl2 to induce WT1

expression. After 24 hours, cells were rinsed with PBS, fixed with 3.7% w/v
paraformaldehyde (Sigma-Aldritch, St. Louis, MO), rinsed with PBS, and permeablized

in 0.5% Triton X-100 (Sigma). Nonspecific immunoglobulin binding was blocked with

5% normal goat serum and 0.5% NP-40 (both from Sigma). Primary antibodies

recognizing -catenin, and E-cadherin (both from BD Pharmingen, San Diego, CA,

catalog numbers 610153 and 610181) were diluted 1:100 in blocking solution. After

incubation with primary antibody, cells were rinsed with 0.05% Tween-20 (BioRad

Laboratories, Hercules, CA) in PBS, and then incubated with fluorescein-conjugated

goat-anti-mouse secondary antibodies (Molecular Probes, Inc., Eugene, OR) diluted to 5

µg/µL in blocking solution. Nuclei were counterstained with DAPI (Roche, Indianapolis,

IN) at a dilution of 1 µg/µL in blocking solution at 37C for a maximum of 2 minutes.

Slides were fixed with one droplet of Anti-Fade reagent (Molecular Probes) before

mounting with a coverslip.



Western Blotting.

Total cellular protein was obtained by harvesting cells in 1X LDS sample buffer

(Invitrogen). Electrophoresis and blotting was performed using reduced samples and the

Invitrogen XCell II SureLock blot system according to manufacturer’s specifications.

Samples were run on 4-12% bis-tris gels using MOPS buffer, blotted onto PVDF

membranes, and blocked overnight at 4C in 5% nonfat milk in TBS (Quality Biological,

Gaithersburg, MD) + 0.05% Tween 20 (BioRad). Primary antibodies were diluted into

blocking solution as follows: WT1 (Santa Cruz, SC-192) 1:1000; p21 (BD Pharmingen,

556430) 1:500; tubulin (Sigma T9026) 1:10,000; Vimentin (Santa Cruz, SC 5565) 1:200;

E-cadherin (BD Pharmingen 610182 ) 1:2500; -catenin (BD Pharmingen 610154)
1:500. Antibody binding was detected with enhanced chemiluminescence (RPN2106,

Amersham Biosciences, Piscataway, NJ).



Proliferation, Cell Cycle Analysis, and Morphology.

MTT Assays. MTT proliferation assays were performed according to manufacturer’s

instructions (Chemicon, Temecula, CA). Cells were seeded at 104 cells/well on 96-well

plates on Day 0. Plates were read on a Vmax kinetic microplate reader (Molecular

Devices, Sunnyvale, CA) at a wavelength of 650 nm.



Cell Cycle Analysis. Cells were seeded at 106 cells per plate on 100 mm plates. Cells

were harvested in “Magic Solution” (0.6% NP-40, 3.7% formaldehyde, 11 g/mL

Hoechst 33258 (Molecular Probes) in PBS), and absorbance quantified using a

FACScalibur (Becton Dickinson) and CellQuest Pro software (Becton Dickinson).



Matrigel. Matrigel Basement Membrane (BD Discovery Labware) was diluted 1:3 with

OptiMEM IX reduced serum media (Invitrogen) with and without 75 µM ZnCl2. Each 35

mm culture plate was seeded in duplicate with 105 cells diluted in 3 mL Matrigel

solution. Plates were incubated at 37C for 30 minutes, after which time 1 mL room

temperature MEGM culture media (Clonetics) was added to each plate. The plates were

then incubated at 37C for 10 days.
Gene Expression Profiling

RNA was obtained from a control cell line (transfected with the empty vector containing

the metallothionein promoter), two independently derived cell lines expressing WT1 (-

Ex5/-KTS) under the control of the metallothionein promoter, and two independently

derived cell lines expressing WT1 (+Ex5/+KTS) under the control of the same promoter

each treated with or without ZnCl2 overnight. RNA was also obtained from one cell line

constitutively expressing WT1 (-Ex5/-KTS). This material was used to probe a cDNA

array (OHS-802, SuperArray Bioscience Corporation, Frederick, MD) containing 440

cancer-related genes. Reverse transcription, array hybridization, and data production

were performed by SuperArray.



Polymerase Chain Reaction

Semiquantitative RT-PCR. RNA was harvested from cells and reverse transcribed as

previously described. The sequences of the primers used to detect B-raf cDNA were as

follows: 5’-GAAGACCTCACAGTAAAAATAGGTGA-3’ and 5’-

CCACAAAATGGATCCAGACA-3’. PCR was performed with a melting temperature

of 95C, followed by annealing at 56C for 60s, and then extension at 72C for 45s for a

total of 30 cycles. PCR products were resolved by electropheresis on a 2% agarose gel.

Conditions and primers for WT1 and 36B4 RT-PCR have been previously described

(Loeb et al., 2001; Loeb et al., 2003).



Quantitative RT-PCR. Quantification of RPS6K gene expression was performed using a

Bio-Rad MyiQ single-color real-time PCR detection system with SYBR green chemistry
in 96 well plates. Primers specific to RPS6K and to the housekeeping gene GAPDH

were ordered from SuperArray Bioscience (Fredrick, MD). Reactions were set up in

triplicate each in a final volume of 25 μl with 12.5 μl SYBR green master mix (Bio-

Rad), 1 μl cDNA, and 400 nM of each primer. Amplification was performed with the

following thermocycler parameters: 95˚C for ten minutes followed by 40 cycles of 95˚C

for 15 seconds and 60˚C for one minute. The mean threshold cycle (Ct) of the triplicate

samples was determined and then the RPS6K Ct for each sample was corrected against

the Ct level of GAPDH. Quantification of gene expression was performed by calculating

ΔΔCt where ΔΔCt = (Ctsample – CtGAPDH)control – (Ctsample – CtGAPDH)treated. The fold change

in gene expression between two samples was then determined by calculating 2-ΔΔCt.
Table 1: Gene Expression Changes Caused by WT1 Induction

          Gene             UniGene Number             Isoform   Up or Down
          BCL2                Hs.150749                 Both        Up
          MDR1                Hs.489033                 Both        Up
          B-Raf               Hs.490366                 Both        Up
        Cyclin D1             Hs.523852                 Both        Up
        Cyclin D2             Hs.376071                 Both        Up
         CRHR1                Hs.417628                 Both        Up
        -catenin             Hs.476018                 Both        Up
          E2F 3               Hs.269408                 Both        Up
          ErbB4               Hs.390729                 Both        Up
         ICAM 1               Hs.515126                 Both        Up
           c-Kit              Hs.479754                 Both        Up
          MMP1                Hs.83169                  Both        Up
         MMP14                 Hs.2399                  Both        Up
         MMP17                Hs.159581                 Both        Up
          L-Myc               Hs.437922                 Both        Up
          N-Myc               Hs.25960                  Both        Up
  PDGF Receptor  chain       Hs.74615                  Both        Up
       Pleiotrophin           Hs.371249                 Both        Up
         Albumin              Hs.418167             (-Ex5/-KTS)     Up
            Bax               Hs.159428            (+Ex5/+KTS)      Up
          EGR1                Hs.326035            (+Ex5/+KTS)      Up
          ErbB3               Hs.118681            (+Ex5/+KTS)      Up
Hyaluronoglucosaminidase 1    Hs.75619             (+Ex5/+KTS)      Up
       Iduronidase            Hs.529517            (+Ex5/+KTS)      Up
            Ret               Hs.350321            (+Ex5/+KTS)      Up
   Ribosomal S6 kinase        Hs.463642            (+Ex5/+KTS)      Up
          VEGF                Hs.73793             (+Ex5/+KTS)      Up
          ETV1                Hs.22634                  Both      Down
          ETV3                Hs.352672                 Both      Down
        Sprouty 2             Hs.18676                  Both      Down
          TGF-a               Hs.170009             (-Ex5/-KTS)   Down
HIV-1 Rev Binding Protein     Hs.443507             (-Ex5/-KTS)   Down
         MNDA                 Hs.153837            (+Ex5/+KTS)    Down
        SLC16A1               Hs.75231             (+Ex5/+KTS)    Down

Listed are select genes that are either up- or downregulated by one or both WT1 isoforms
as indicated. Fold induction is not included on the table because for many genes, there
was little or no detectable expression in control cells, making calculation of induction
ratio impossible.