Transcription induced by EGFR activation often involves the coordinated expression of genes that positively and negatively regulate the original signaling pathway; therefore, altera-tion
Trang 1associated with MIG6 expression
Takeshi Nagashima1, Ryoko Ushikoshi-Nakayama1, Atsushi Suenaga2, Kaori Ide1, Noriko Yumoto1, Yoshimi Naruo3, Kaoru Takahashi1, Yuko Saeki1, Makoto Taiji2, Hiroshi Tanaka3, Shih-Feng Tsai4 and Mariko Hatakeyama1
1 Cellular Systems Modeling Team, Computational Systems Biology Research Group, Advanced Computational Sciences Department, RIKEN Advanced Science Institute, Yokohama, Kanagawa, Japan
2 High-Performance Molecular Simulation Team, Computational Systems Biology Research Group, Advanced Computational Sciences Department, RIKEN Advanced Science Institute, Yokohama, Kanagawa, Japan
3 School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan
4 Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan
Keywords
EGFR; gene expression; MIG6; mutation;
signal transduction
Correspondence
M Hatakeyama, Cellular Systems Modeling
Team, Computational Systems Biology
Research Group, Advanced Computational
Sciences Department, RIKEN Advanced
Science Institute, 1-7-22 Suehiro-cho,
Tsurumi-ku, Yokohama, Kanagawa
230-0045, Japan
Fax: +81 45 509 9613
Tel: +81 45 509 9302
E-mail: marikoh@gsc.riken.jp
Database
Microarray data used in the present
study have been deposited in the Gene
Expression Omnibus database (http://www.
ncbi.nlm.nih.gov/geo/) with accession
number GSE11729
(Received 10 May 2009, revised 14 July
2009, accepted 16 July 2009)
doi:10.1111/j.1742-4658.2009.07220.x
Controlled activation of epidermal growth factor receptor (EGFR) is sys-tematically guaranteed at the molecular level; however, aberrant activation
of EGFR is frequently found in cancer Transcription induced by EGFR activation often involves the coordinated expression of genes that positively and negatively regulate the original signaling pathway; therefore, altera-tions in EGFR kinase activity may reflect changes in gene expression asso-ciated with the pathway In the present study, we investigated transcriptional changes after EGF stimulation with or without the EGFR kinase inhibitor Iressa in H1299 human non-small-cell lung cancer cells [parental H1299, H1299 cells that overexpress wild-type EGFR (EGFR-WT) and mutant H1299 cells that overexpress EGFR where Leu858 is substituted with Arg (L858R)] The results obtained clearly demonstrate differences in transcriptional activity in the absence or presence of EGFR kinase activity, with genes sharing the same molecular functions showing distinct expression dynamics The results show the particular enrichment of EGFR⁄ ErbB signaling-related genes in a differentially expressed gene set, and significant protein expression of MIG6⁄ RALT(ERRFI1), an EGFR negative regulator, was confirmed in L858R High MIG6 protein expres-sion was correlated with basal EGFR phosphorylation and inversely corre-lated with EGF-induced extracellular signal-regucorre-lated protein kinase phosphorylation levels Investigation of the NCI-60 cell lines showed that ERRFI1expression was correlated with EGFR expression, regardless of tis-sue type These results suggest that cells accumulate MIG6 as an inherent negative regulator to suppress excess EGFR activity when basal EGFR kinase activity is considerably high Taking all the above together, an EGFR mutation can cause transcriptional changes to accommodate the activation potency of the original signaling pathway at the cellular level
Abbreviations
AU, approximate unbiased; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated protein kinase; GEO, gene
expression omnibus; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NSCLC, human non-small-cell lung cancer; SHC, Src homology 2 domain containing.
Trang 2Epidermal growth factor receptor (EGFR) is a
mem-brane tyrosine kinase that is involved in the regulation
of a wide variety of biological processes [1] Controlled
activation of EGFR is systemically and evolutionarily
guaranteed by the presence of a variety of ligands, or
by dimerization and trans-activation with other family
member receptors of ErbB [2,3] Additionally, the
potency and dynamics of EGFR signaling are
adap-tively tuned via biochemical parameters such as affinity
constants, catalytic activities, the concentration of the
signaling mediators and plastic pathway architectures
[4–6], thereby ensuring that cells can produce the
desired outputs in response to various cellular
condi-tions Transcription induced shortly after ligand
stimu-lation is quantitatively regulated by upstream signaling
dynamics, which concomitantly regulate the dynamics
of the original pathway Kinases and effector proteins
in the EGFR-mitogen-activated protein kinase
(MAPK) cascade are particular major targets [7–10]
Therefore, quantitative transcriptional outcomes, in
addition to qualitative ones, may be altered if EGFR
kinase activity is modified by mutation,
overexpres-sion, or suppressed by inhibitors such as Iressa
(Gefiti-nib, AstraZeneca, London, UK) or Tarceva (Erloti(Gefiti-nib,
Roche, Basel, Switzerland)
In the present study, time-course genome-wide gene
expression was investigated, aiming to delineate the
transcriptional outcomes induced by EGFR activation
under various conditions In brief, the human
non-small-cell lung cancer (NSCLC) cell line H1299 with
EGFR overexpression (wild-type: EGFR-WT) and
with or without the mutation in which Leu858 is
substituted with Arg (mutant: L858R), in addition to
the parental cell line, was used Various point
muta-tions at L858R, L861, S768, E709 and G719 in the
EGFR kinase domain, insertions in exon 20 and
dele-tion mutadele-tions in exon 19 of the gene for EGFR are
often found in NSCLC patients Among these, the
L858R mutation has been known to be a good
predic-tive marker of Iressa (Gefitinib) responsiveness [11–13]
Therefore, delineating the transcriptional regulation of
this mutant is of clinical importance in terms of
con-tributing towards our understanding of patient
sensi-tivity to Iressa, as well as side effects and drug
resistance The results obtained demonstrate
differ-ences in EGF-stimulated transcription in the absence
or presence of Iressa in all cell lines tested, and show
that the expression dynamics of the affected genes with
overlapping molecular functions are distinct in each
cell group Particular enrichment of cell-specific genes
involved in the cell cycle and MAPK signaling
path-way was found and, of these, we confirmed significant protein expression of the EGFR negative regulator MIG6⁄ RALT only in L858R cells
MIG6⁄ RALT is known to be a transcriptional feed-back regulator of the ErbB-MAPK signaling pathway [14,15] and its loss is associated with ErbB2⁄ HER-2 oncogenic potency leading to Herceptin resistance [16] Furthermore, its overexpression is associated with down-regulation of phosphorylated-ErbB2 [17] in breast cancer The present study, using lung cancer cell lines with various EGFR mutants, suggested that endogenous MIG6 may be directly associated with basal EGFR kinase activity Cells might accumulate MIG6 to suppress excess EGFR activity; therefore, MIG6 may be regarded as a molecular marker for indicating the intrinsic kinase activity of EGFR, regardless of tissue type
Results
Ligand-induced transcriptional signatures of EGFR-WT and L858R in the absence or presence
of EGFR kinase activity The transcriptional activity that follows EGFR activa-tion often involves the expression of genes that nega-tively and positively adjust the magnitude and duration of the original EGFR-MAPK signaling path-ways [7–10,18] Therefore, quantitative transcriptional outcomes, in addition to qualitative ones, may be altered if EGFR kinase activity is modified by muta-tion and overexpression, or suppressed by kinase inhibitors such as Iressa Accordingly, time-course microarray analysis was performed to identify genes that functionally modulate the EGFR signaling path-way For this purpose, three derivatives of human NSCLC cell lines, comprising parental H1299,
EGFR-WT and L858R, were employed as cellular systems The overall transcriptional signatures after EGF administration in the absence or presence of the EGFR kinase inhibitor Iressa were investigated using Affymetrix GeneChip (U133Plus 2.0; Affymetrix, Santa Clara, CA, USA) The workflow of gene expres-sion data analysis is shown in Fig 1
Cell-specific differentially expressed genes and effect of EGFR kinase inhibitor on gene expression
First, the overall gene expression profiles in the EGF- and Iressa-stimulated three cell lines were
Trang 3examined A hierarchical clustering together with
assessment of cluster uncertainty was carried out
according to the expression levels of all probe sets on
the array for each cell stimulated with EGF in the
absence or presence of Iressa Cluster analysis clearly
showed distinct transcriptional outcomes in the three
cell lines Interestingly, the cellular response of L858R
was similar to that of EGFR-WT in terms of the EGF
response, and similar to the parental cell line in the
presence of Iressa (Fig 2A)
Cell-specific gene expression associated with EGFR
activity was determined using two criteria: (a) where
the expression level shifted relative to nonstimulated
cells after stimulation by EGF or EGF + Iressa
(ligand responsive genes) and (b) where the expression
level was altered in the absence or presence of Iressa
(kinase responsive genes) As a result, 746, 1034 and
1444 genes were identified for H1299, EGFR-WT and
L858R, respectively (2234 genes in total) (Fig 2B)
The list of induced genes obtained included DUSP6 (a
MAPK phosphatase), ERBB2 and ERBB3, which
modulate EGFR signaling
Cluster analysis of selected genes clearly showed a
discrepancy in the expression dynamics of each cell
type (Fig 2C) Although H1299 only had two major
clusters (simple ascending and descending), EGFR-WT
and L858R cells showed multiple clusters for EGF or
EGF + Iressa stimulation Additionally, comparison
of EGF- and EGF + Iressa-induced time courses
highlighted differences in the effect of Iressa on gene expression dynamics in the three cell lines (Fig 3) Consistent with the clustering results, Iressa induced only minor changes in gene expression of the parental cell line (Fig 3A) On the other hand, EGFR-WT exhibited larger changes in gene expression levels in response to Iressa compared to L858R, whereas a lar-ger change in the expression time course was observed for L858R (Fig 3B) Furthermore, our analysis revealed a distinct time-dependent effect of Iressa among the cell lines examined (Fig 3C) In
EGFR-WT, the effect of Iressa on gene expression was rather temporal (4–6 h), whereas its effect was more persis-tent in L858R (> 10 h)
Different transcriptional regulation of biological pathways induced by ligand and EGFR kinase inhibitor
In an effort to assess the biological functions of the differentially expressed genes described above, func-tional enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway [19] and Gene Ontology (GO) [20] databases The results showed that EGF and Iressa altered the expression levels of genes involved in specific biological processes, such as the cell cycle, circadian rhythm, MAPK signaling pathway, small cell lung cancer and p53 signaling pathway (Table 1) Furthermore, GO term analysis for individual clusters highlighted the commonality and discrepancy of cellular responses to ligand stimulation in the absence or presence of EGFR kinase activity (Table S1) For example, genes involved
in transcriptional regulation and protein binding were found to be enriched in the early response gene cluster
of the three cell lines (clusters presented within a red bar in Fig 2C) Genes associated with cell cycle func-tions were also significantly selected for all cell lines; however, the time-course expression patterns differed for each Different expression time courses for the same molecular function were also observed, such as genes related to signal transduction via receptor bind-ing and receptor activity Thus, a difference in EGFR activity can result in the distinct transcriptional regula-tion of important biological processes that may con-tribute to the sensitivity of the cells to Iressa or ligand
Identification of direct EGFR regulators through functional annotation of Iressa-induced
differentially expressed genes
As described above, EGF and Iressa-induced overall expression dynamics differed between cell lines, and
Fig 1 Workflow of gene expression data analysis The workflow
of microarray data analysis applied in the present study is shown.
Trang 4these differences were observed even in those genes
associated with same molecular functions In an effort
to further examine how a single amino acid mutation
in the EGFR kinase domain affects downstream gene
expression, those genes likely to be under the
regula-tion of activated EGFR were examined Genes
show-ing distinct time-course patterns in the absence or
presence of Iressa were extracted (a correlation
coeffi-cient of <)0.5 between EGF and EGF + Iressa) As
a result, 405 genes were selected (12, 117 and 299 genes for H1299, EGFR-WT and L858R, respectively) Of these, KEGG pathway analysis revealed that the ErbB signaling pathway is enriched in L858R (P = 0.02057; Bonferonni corrected) Furthermore, functional annotation using public databases identified 52 out
of 405 genes (12.8%) as comprising regulators of EGFR⁄ ErbB and MAPK signaling pathways (Fig 4A)
A
B
C
Fig 2 Gene expression profiles in H1299, EGFR-WT and L858R after EGF or Iressa stimulation (A) Overall similarity of gene expression profiles in EGF- and Iressa-stimulated cells Clusters with high AU values (> 95) are highlighted by red rectangles (B) The number of differ-entially expressed genes in each cell line The Venn diagram represents the number of differdiffer-entially expressed genes obtained using two selection criteria (see Experimental procedures) in H1299, EGFR-WT and L858R cell lines Numbers in round and square brackets represent the number of probe sets and the number of probe sets without gene IDs, respectively Other numbers refer to the number of genes (C) Clustering of expression profiles of differentially expressed genes Representative genes were shown by number: 1, ERRFI1; 2, DUSP6; 3, SPRY4; 4, ERBB3; 5, ERBB2 Ctrl, control (no stimulation); I, Iressa; E, EGF; E+I, EGF and Iressa.
Trang 5Among these, we noted cell-specific differential
expression of two direct EGFR regulators: SPRY4
(Sprouty-4) and ERRFI1 (MIG6)
Sprouty family member proteins are known to be
negative and positive regulators of fibroblast growth
factor and EGFRs, respectively [10,21–23] In our
analysis, SPRY4 expression was stimulated by EGF and reduced by the addition of Iressa in EGFR-WT How-ever, the induction of Sprouty-4 remained unchanged at the protein level in both cell types (data not shown) MIG6 (RALT or ERRFI1) is a cytoplasmic adapter protein that can inhibit EGFR kinase activity through
B
Fig 3 Iressa-induced differences in gene expression Iressa-induced differences in gene expression amplitude (A) and time course (B) Dif-ferences in Iressa-induced gene expression were calculated using two indexes: (a) the Icvalue that reflects differences in the expression level and (b) the correlation coefficient which represents differences in the expression pattern Two time-course profiles (EGF and EGF + Iressa) of selected genes were used for the analysis The distribution of I c and correlation coefficient in three cell lines are shown in (A) and (B), respectively Larger (Ic> 10) and smaller (Ic< )10) I c values were rounded to 10 and )10, respectively (C) The number of probe sets where the expression level was altered by Iressa at individual time points in H1299, EGFR-WT and L858R.
Trang 6direct binding to the kinase domain [15,24–26]
ERR-FI1expression was significantly induced in EGFR-WT
and L858R cells Although ERRFI1 was reduced in
response to EGF stimulation in H1299 cells, it was
transiently expressed in EGFR-WT cells and
constitu-tively expressed in L858R cells Western blot analysis
identified higher basal levels of MIG6 protein in
L858R compared to parental H1299 and EGFR-WT
cells, and little change was observed in response to
EGF stimulation (Fig 4B)
The evolutionarily conserved ErbB-binding region
(EBR) of MIG6 is known to bind the RYLVIQ
sequence of EGFR (amino acids 953–958), which
par-ticipates in the allosteric control of EGFR activity
[26] Therefore, high expression of MIG6 may be able
to suppress the effect of EGF for EGFR
phosphoryla-tion in EGFR-WT Indeed, our validaphosphoryla-tion experiment
confirmed that overexpression of MIG6 decreased the
phosphorylation of EGFR in the presence of a high
concentration of EGF in EGFR-WT cells (data not
shown), as previously reported for breast [17] and
other cell lines [24]
Accordingly, the expression level of MIG6 should be
associated with high EGFR expression levels because
the overexpression of EGFR has often been linked to
high EGFR kinase activity Therefore, ERRFI1
expres-sion in various cancer cell lines was investigated using
the publicly available NCI-60 dataset The dataset was
downloaded from the Gene Expression Omnibus (GEO)
database (http://www.ncbi.nlm.nih.gov/geo/; accession
number = GSE5720) The results of the analysis
sur-prisingly showed that ERRFI1 expression levels varied
among all cell lines and no tissue-specific trend was
observed (Fig 5A), although a previous study reported
tissue-specific expression of ERRFI1 in some cancers
[24] ERRFI1 expression was most correlated with
EGFR expression, regardless of cancer cell type, and
was not correlated with other ERBB gene expression
(Fig 5B) The results indicate that MIG6 could be uti-lized as a molecular marker for indicating the func-tional activity of EGFR in tissues, regardless of cancer type Indeed, our transcriptional analysis indicated that Iressa totally abolished the expression of ERRFI1 in EGFR-WT and L858R cells (Fig 2C) Accordingly, ERRFI1may operate as a molecular sensor to monitor EGFR kinase activity
Relationship between MIG6 expression and EGFR mutation
Although a functional role of MIG6 in relation to EGFR kinase regulation has been reported, as described above, and was confirmed in the present study, its relationship to Iressa sensitivity, EGFR mutation and the MAPK signaling pathway has not been reported
Earlier studies found that clinical responsiveness to Iressa was closely associated with EGFR mutations such as L858R and delL747-P753insS in the kinase domain, which also enhance EGF-dependent EGFR activation [11,12] Huang et al [13] performed muta-tional analysis of the EGFR gene from exons 18–21 in
a series of surgically resected NSCLCs and found a high mutation rate for EGFR in Taiwanese patients
In addition to major mutation types such as the L858R mutation and deletions in exon 19, various point mutations at residues L861, S768, E709, G719 and H835 and insertions in exon 20 of the EGFR gene have been observed [13]
Accordingly, other H1299 derivatives that overex-press different types of EGFR mutants, including EGFR-Del (deletion of the kinase domain), S768I, L861Q, E709G and G719S [27], in addition to parental H1299, EGFR-WT and L858R, were assessed by quan-titative western blot analysis in an effort to delineate the relationship between EGFR mutation, Iressa sensitivity
Table 1 Enriched KEGG pathways in differentially expressed genes.
KEGG pathway
*P < 0.01, **P < 0.05.
Trang 7and MIG6 expression Surprisingly, MIG6 expression
was significantly high in L861Q and G719S cells
Accordingly, no clear correlation was observed between
MIG6 expression and Iressa sensitivity in the eight cell
lines tested (Figs 6A and S1A) However, MIG6
expres-sion levels were uniquely correlated or anti-correlated
with the phosphorylation state of EGFR, Src homology
2 domain containing (SHC), mitogen-activated protein
kinase kinase (MEK) and extracellular signal-regulated
protein kinase (ERK) in the absence or presence of
EGF (Figs 6B and S1B) Interestingly, MIG6 expression
showed good correlation with basal phosphorylation
levels of EGFR (correlation coefficient = 0.61) and
with its direct effector protein SHC (correlation
coefficient = 0.75) in the absence of stimuli, and strong
anti-correlation (correlation coefficient =)0.83) with
ERK phosphorylation in the presence of stimuli These
results imply that increased MIG6 expression effectively
inhibits signal transduction to the downstream pathway
when EGFR is irregularly activated
Discussion
In the present study, we investigated the property of biological networks under various conditions related to EGFR kinase activity, which was altered by single amino acid mutation, activation by EGF and suppres-sion by Iressa Time-course microarray analysis enabled us to identify differentially expressed genes and obtain insight into the dynamic behavior of coor-dinated transcription associated with their upstream signaling pathways and functions
The L858R mutation of EGFR has been shown to
be a good predictive marker in terms of Iressa treat-ment [11,12] The data obtained in the present study showed that Iressa effectively suppressed EGF-induced expression of DUSP6 and ERRFI1 and, at the same time, increased the expression of ERBB2 and ERBB3 (i.e dimerization partners of EGFR) in L858R cells The regulatory pattern of these four genes suggests that the activation of EGFR or ErbB2-3 pathways is an
Fig 4 Direct EGFR regulator ERRFI1
(MIG6) is included in the Iressa responsive
MAPK and ErbB signaling-related gene list,
its protein expression (A) 52 MAPK and
ErbB signaling-related genes in 405 Iressa
responsive genes are shown Genes
involved in the EGFR ⁄ ErbB signaling
path-way are highlighted in blue Genes included
in the 405 gene group, which were
differen-tially expressed and where the expression
pattern was reversed by Iressa, are shown
in red Genes included in 2234 gene group,
but not in the 405 gene group, are depicted
in orange PG, PubMed and GeneRIF.
(B) MIG6 protein expression in EGF (10 n M )
or EGF + Iressa (10 l M ) stimulated H1299,
EGFR-WT and L858R as determined by
western blot analysis The experiment was
performed twice independently.
Trang 8immediate transcriptional effect of Iressa Given that
L858R cells are more sensitive to Iressa [27], inhibition
of EGFR kinase activity may lead to the activation of
alternative pathways that compensate for the loss of
EGFR pathway activity in L858R cells Indeed,
ERR-FI1demonstrated an anti-correlated expression pattern
with ERBB2 and ERBB3 in various tumors and cancer
cell lines [15,16], and higher expression levels of
recep-tor tyrosine kinase genes were observed in other
NSCLC cell lines showing high Iressa sensitivity (data
not shown) Thus, the inherited molecular fragility of
L858R in terms of Iressa sensitivity appeared to be
neutralized by transcriptional feedback
Although we initially speculated that MIG6
expres-sion was EGF-inducible, as was observed for ERRFI1
expression, this was not the case Rather, we found that
MIG6 expression was static and correlated with basal
phosphorylation levels of EGFR and SHC, and was
negatively correlated with EGF-stimulated
phosphory-lated ERK levels in H1299 cell lines The presence of
high levels of MIG6 expression might ensure, in the eight cell lines examined in the present study, that sig-nal transduction downstream of EGFR is disturbed However, further investigations are required to eluci-date the regulatory mechanism of MIG6 in relation to the EGFR mutation
The present study is the first to show the association
of MIG6 expression with the EGFR mutation in cancer MIG6⁄ RALT is known to be a transcriptional negative regulator of EGFR signaling [14] Ferby et al [24] also showed: (a) reduced expression of ERRFI1 in skin, breast, pancreatic and ovarian cancers, as well as psoriasis; (b) an inverse relationship between MIG6 expression and phosphorylated EGFR; and (c) an inverse correlation between ERRFI1 and ERBB3 mRNA levels in human melanoma cell lines Their results suggest that down-regulation of MIG6 in tumors and cancer cell lines leads to activation of ErbB signaling [24] Although the results obtained in the present study partially support those reported in
A
Fig 5 ERRFI1 and ErbB receptor family member expression in NCI-60 (A) ERRFI1 gene expression level in various cancer cell lines The dataset comprises nine different types of cancer, presented in different colors Vertical and horizontal axes represent cell line names and the gene expression level, respectively (B) Cluster analysis of ERRFI1 and ERBB expression in the NCI-60 dataset Prior to cluster analysis, the expression level of a gene was normalized so that the mean = 0 and SD = 1 Red and blue represent high and low normalized expression levels, respectively Color bars at the top of the heatmap represent the cancer type, with the same colors being used in the upper panel.
Trang 9earlier studies [24], our data clearly showed that MIG6
expression is correlated with the phosphorylated active
state of EGFR and that ERRFI1 expression is
associ-ated with basal EGFR kinase activity in the absence
of ligand Furthermore, MIG6 expression may be
indi-rectly (i.e not diindi-rectly) associated with EGFR or other
ERBB mRNA levels Among the ErbB receptor
fam-ily, the ErbB2 receptor is the most preferred binding
partner that leads to activation of EGFR kinase
[28,29] Thus, cells with distinct EGFR mutations have
their total signaling activity modulated at the
molecu-lar (kinase activity) and transcriptional levels, and
these modulations might compensate each other to
control the final cellular output at the systems level
Experimental procedures
Cell culture and RNA isolation
EGFR-mutated H1299 human lung cancer derivatives
were established as described previously [27] Cells were
maintained in RPMI medium supplemented with 10%
fetal bovine serum and 1 mm sodium pyruvate Prior to growth hormone treatment, cells were serum-starved for 16–24 h For EGFR kinase inhibition, Iressa (a generous gift from Astra Zeneca, London, UK) was added 20 min prior to growth hormone administration For the tran-scriptional analysis, cells were incubated with 10 nm EGF for 0.5, 1, 2, 4, 6 or 10 h and then washed twice with NaCl⁄ Pi Cells not treated with growth hormone were used as the control Cells were scraped using ice-cold NaCl⁄ Pi containing 10 lgÆmL)1 cycloheximide Total RNA was isolated using TRIzol reagent (Life Technolo-gies Corporation, Carlsbad, CA, USA) and then purified using the QIAGEN RNeasy Mini kit RNA quality was assessed using a Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA)
Western blot analysis Cells were treated with EGF in the absence or presence of Iressa for the indicated time period, washed three times with NaCl⁄ Pi, and then lysed using Bio-Plex lysis buffer (Bio-Rad laboratories, Hercules, CA, USA) The cell
B
Fig 5 Continued.
Trang 10A B
Fig 6 Comparison of MIG6 expression and phosphorylation of signaling molecules (A) MIG6 expression levels in eight H1299 derivative cell lines was compared with the Iressa sensitivity reported in a previous study [22] IC50values were extracted from fig 2b,c of the same study [22] and values < 0.02 were rounded to 0 (B) Basal MIG6 protein expression levels were compared with phosphorylation levels of EGFR, SHC, MEK and ERK in eight H1299 derivative cell lines in the absence (left) or presence (right) of EGF stimulation (5 min, 10 n M ) Protein expression and phosphorylation were measured by western blot analysis Western blot images are shown in Fig S1 Horizontal and vertical axes represent quantified signal intensities for MIG6 and signaling molecules (EGFR, SHC, MEK and ERK), respectively.