SUPPLEMENTARY MATERIALS AND METHODS
Cell proliferation assay for HCC4006 and HCC4006TGFβ1 cells
HCC4006 cells were cultured in the presence of 10ng/ml TGFβ1 for more than 30
days prior to the assay. The resulting HCC4006TGFβ1 cells were confirmed
mesenchymal using Western blot. Exponentially growing and viable HCC4006 and
HCC4006TGFβ1 cells were seeded at 9.0 X 105 cells/ml in 10cm cell culture dish.
HCC4006 cells were cultured in RPM1640 supplemented with 10% FBS and
HCC4006TGFβ1 cells were cultured in RPM1640 supplemented with 10% FBS and
10ng/ml TGFβ1 for 5 days. At the end of the assay, cells were trypsinized and viable cells
were counted using Countess (Life) with trypan blue exclusion method. Three
independent assays were performed.
STR assay
DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). DNA
samples
were
first
quantified
using
NanoDrop
ND-2000
Spectrophotometer
(ThermoScientific) then the concentration of DNA was measured again with Qubit (Life).
Samples were submitted to Dana-Farber Cancer Institute Shannon McCormack
Molecular Diagnostic Laboratory or University of Illinois at Chicago Center for Genomic
Research–DNA Services Facility or Genetica DNA Laboratories.
List of Antibodies
Target
Use
Vendor
Catalog Number
EGFR
EGFR (Y1068)
EGFR (Y1173)
MET
Akt
Akt (S473)
Erk
WB
WB
IHC
WB
WB
WB
WB
Santa Cruz
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
SC-03
3777
4407
4560
4685
4060
9102
1
Erk (T202/Y204)
SMAD2
SMAD2 (S465/4467)
actin
E-cadherin
N-cadherin
Vimentin
tubulin
S6
S6 (S240/Y244)
ERBB2 (HER2)
ERBB3 (HER3)
TGFBR1
TGFBR2
Bim
Actin
SMAD2(S465/467)/
SMAD3 (S423/425)
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
IF
IF, IHC
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
Cell Signaling Tech
9101
5339
3108
4967
3195
4061
5741
2128
2217
2215
2165
4754
3712
3713
2933
3700
9510/8828
(9510, IHC)
Luminex Assay
Quantification of TGF-β isoforms was assayed using Human TGF-β 1,2,3 multiplex
assay (R&D Systems) or using Human TGF- β1 single-plex assay (Life). Growth factors
and cytokines were measured using Cancer Growth Factor 12-plex panel (Millipore,
discontinued) and Human Cytokine 30-plex panel (Life). Cells were seeded in 60mm
dishes so that the cells were 70% confluent on the day of assay. Cells were treated with
drugs for indicated time and supernatant was harvested. If no drugs were used, then the
cells were cultured for indicated amount of time and supernatant was harvested. Cell
debris was removed from the supernatant by centrifugation, snap-frozen in LN2, and kept
in -80°C freezer until use. The volume of supernatant and the number of the cells or the
amount of protein upon cell lysis were recorded at the time of the harvest to normalize
the amount of targets. To measure phosphorylated SMAD 2 and 3, we used MILLIPLEX
MAP TGFβ Signaling Pathway Magnetic Bead 6-Plex - Cell Signaling Multiplex Assay
2
(48-614MAG, Millipore). Assays were performed according to the manufacturer's
specifications and analyzed with a Luminex FM3D platform. For SMAD2/3 assay, activity
was expressed in mean fluorescence intensity (MFI) with background correction from
duplicate samples. Statistical significance was calculated using a two-sided Student's
exact t-test.
shRNA knockdown and sequence used for study
shRNA knockdown and viral transduction and siRNA transfection Lentiviral
production and infection were performed as reported previously (2). Briefly, the 293LTV
cell line (Cell Biolabs) was transfected with pLKO.1 constructs and packaging plasmids,
pCMV-ΔR8.2dvpr and pCMV-VSV-G. Lentiviral supernatants were collected and applied
to target cells. Following puromycin selection, cell viability assays were performed 7 days
post-infection. Quality of viral supernatant was routinely tested with Lenti-X Go Stix
(Clontech).
Target
The RNAi
number
consortium
sequence
Remarks
EGFR
TRCN0000010329
5’ – AGAATGTGGAATACCTAAGG – 3’
NonTarget
N/A
5’ – GCGCGATAGCGCTAATAATTT – 3’
Sigma SHC-002
DNA sequencing
The PCR primers used for the amplification of the EGFR exons 19 and 20 were
the following: EGFREX19-F: 5’-GTGGCACCATCTCACAATTGCC-3’’ EGFREX19-R: 5’GGGCCTGAGGTTCAGAGCCAT-3’ which generate a 203 bp amplicon; EGFREX20-F:
5’-ATGCGTCTTCACCTGGAAGG-3’
and
EGFREX20-R:
5’-
CGCAGACCGCATGTGAGGAT-3’ (337bp amplicon). PCR reactions were performed
using the JumpStart Taq polymerase (Sigma) using an annealing temperature of 62 oC.
3
Analysis of Microarray Gene Expression Data
Hybridization to Human Genome U133A v2.0 chips was performed following
Affymetrix protocols (Affymetrix, Inc.). Gene expression data were normalized using the
Robust
Multi-array
(RMA)
method
from
Genepattern
suite
(http://www.broadinstitute.org/cancer/software/genepattern). After normalization, probes
representing
the
same
genes
were
collapsed
into
a
single
value.
GSEA (http://www.broadinstitute.org/gsea) was used to determine the gene set
enrichment of EMT-related, TGFβ, EGFR TKI resistance, and mesenchymal cell
signatures obtained from MsigDB (http://www.broadinstitute.org/gsea/msigdb). GSEA
estimates whether the members of a given gene signature are found at the top or bottom
of a list of genes ranked by signal-to-noise ratio, indicating they are associated with a
specific phenotype (EGFR TKI resistance), rather than being distributed uniformly or
randomly across the gene list. An enrichment score (ES) is calculated to quantify the
degree to which a gene signature is overrepresented at the top or bottom of the entire
ranked list. GSEA normalizes the ES for each gene set to account for the variation in set
sizes, yielding a normalized enrichment score (NES) and a false discovery rate (FDR).
The FDR gives an estimate of the probability that a set with a given NES represents a
false positive finding; it is computed by comparing the tails of the observed and empirical
phenotype-based permutated null distributions for the NES. The original expression data
has been submitted to Gene Expression Omnibus (GEO).
4
Summary of STR Profiling
5