Supplementary methods.
Organotypic Invasion Assay and Immunohistochemistry
The organotypic invasion assay is described eleswhere (Edward et al., 2005).
Briefly, ~7.5x104/ml primary human fibroblasts were embedded in a threedimensional matrix of rat-tail collagen І detached from the culture dish. The
collagen matrix was allowed to contract to ~1.5 cm diameter for approximately
6 days in complete medium (10% FCS/DMEM). Subsequently, 4x104 Scc9 cells
were plated on top of the matrix in complete medium and allowed to grow for 5
days. The matrix was then mounted on a metal grid and raised to the air/liquid
interface resulting in the matrix being fed from below with complete medium
that was changed every 2 days. After 8-12 days, the cultures were fixed in 4%
formaldehyde.
For immunohistochemistry, paraffin embedded tissue was sectioned and stained
with hematoxylin and eosin (H&E) using standard methods. Sections were
labeled with Ki67 using the Dako EnVision rabbit kit (K4011) and
counterstained with hematoxylin.
Immunoprecipitation and western blotting.
For EGFR immunoprecipitations, cells were grown to confluence and
starved overnight with 0.1% normal culture media with or without Ca2+. Cells
were stimulated with 25 ng/ml EGF for 10 minutes and lysed on ice with TNE
buffer (50mM Tris pH 7.5, 140mM NaCl, 5mM EDTA) containing protease and
phosphatase inhibitors. 1 g of EGFR antibody was used to IP from 500 g of
protein for 2 hours at 4 0C followed by 20 l protein G beads for 1 hour. Beads
were washed 3x in lysis buffer and boiled in 2x SDS-PAGE buffer (Invitrogen).
For Western blotting, stimulated cells were lysed and equal amounts of
protein were resolved on 4-12% SDS-PAGE gels (Invitrogen) and blotted to
PVDF membrane using standard methods. Membranes were blocked and probed
over night with the appropriate antibody. Blots were washed and incubated with
appropriate secondary antibody for an hour. Blots were then washed extensively
and developed using SuperSignal West Pico ECL from (Pierce) using a Syngene
Chemigenius gel imaging system.
MTT proliferation assay.
Cells were seeded at 1 x 104 cells per well of a 12-well plate in (4 replicate
samples) and cultured overnight. The following day, cells were switched to 0.1%
normal medium containing 10 ng/ml EGF. For each time point, cells were
incubated with 1 mg/ml methylthiazolyldiphenyl-tetrazolium bromide (MTT)
for 4 hours and the blue formazan product dissolved in DMSO. The absorbance of
samples was measured on a spectrophotometer plate reader using 560 nm filter
and 690 nm filter for a reference wavelength.
For Gefitinib and Erlotinib treatments, 2000 cells were seeded per well of
a 96 well plate. The following day drugs were added at increasing concentrations
and cells incubated for 5 d. Plates were developed as described above. Data was
analyzed in GraphPad Prism and curves were fitted using a sigmoidal doseresponse (variable slope) equation. Significance was calculated using non-linear
regression.
Antibodies
Anti-EGFR for western blotting (1005, Santa Cruz Biotech.), total Akt (cat no.
9272), phosphorylated Akt (cat no. 9271S), total Erk1/2 (cat no. 9102),
phosphorylated Erk1/2 (cat no. 9101S) from Cell Signaling Technology. EGFR for
immunoprecitation (SC-03, Cat no. 555996, BD Pharminogen), anti-Tubulin was
from Sigma-Aldrich (DM1A, cat no. T6199), anti-E-cadherin (cat no. 610182; BD
Biosciences, 1:1000) and anti-Ki67 (cat. no. RM-9106-S, Lab Vision, 1/200).
Mtss1 antibody is described in (Bompard et al., 2005).
Supplementary Figure 1. Mtss1 over-expression does not alter the surface
expression of E-cadherin. Scc9 cells ± Mtss1-GFP were starved overnight in
0.1% growth medium, were stimulated with 25 ng/ml EGF for 0, 10 and 30
minutes and their surface proteins biotinylated. (A) Lyastes were probed for Ecadherin expression. (B) Quantification of E-cadherin expression relative to total
E-cadherin in Scc9 cells ± Mtss1-GFP showing mean ± S.E.M. from 3 independent
experiments.
Supplementary Figure 2. Transient over-expression of myc-Mtss1 affects
EGF-receptor signaling. HeLa cells were transfected with myc-Mtss1, serum
starved overnight and stimulated with 25 ng/ml EGF. Lysates were probed for
EGFR (EGF-R), total Akt (t-Akt) and Mtss1 (A) and the expression levels are
quantified in (B) by densitometry. Mean ± S.E.M. from 3 experiments is shown
relative to Scc9 cells at t = 0 minutes. (C) Lystes were probed for total (tErk1/2)
and phosphorylated Erk1/2 (pErk1/2). (D) Graphs show quantification of
Erk1/2 phosphorylation by densitometry analysis of western blots. Mean density
of bands relative to Scc9 controls at t = 30 minutes ± S.E.M from 3 experiments is
shown. Significance in both (B) and (D) was calculated using an unpaired T-test,
** p < 0.01, * p < 0.05.
Supplementary Figure 3. Increasing cell density reduces Mtss1-enhanced
EGF signaling. (A) Scc9 cells ± Mtss1-GFP were grown to confluence and left for
1-3 days. Lysates were analyzed for basal Erk1/2 and Akt phosphorylation. (B)
Quantification of phospho-Erk1/2 and Akt in Scc9 cells ± Mtss1-GFP. Mean
intensity is shown normalized to tubulin ± S.E.M. from 3 independent
experiments. Significance in (B) was calculated using an unpaired T-test, ** p <
0.01, * p < 0.05. Scc9 cells and Mtss1-GFP expressing cells were grown to
confluence, starved overnight and were then stimulated with 25 ng/ml EGF for
30 minutes and lysates prepared and analyzed for Erk1/2 and Akt
phosphorylation. (C) Mtss1-expressing Scc9 cells were pre-treated with two
rounds of non-targeting (NT) or E-cadherin siRNA and grown to confluence,
starved overnight and stimulated with EGF. Lysates were western blotted for
phospho- and total-Erk1/2 (pErk1/2 and tErk1/2) and phospho- and total-Akt
(pAkt and tAkt). Typical western blots are shown from 3 experiments.