EGFR Tyrosine Kinase Inhibitors Activate Autophagy as a
Cytoprotective Response in Human Lung Cancer Cells
Weidong Han1, Hongming Pan1*, Yan Chen1, Jie Sun2, Yanshan Wang3, Jing Li1, Weiting Ge4, Lifeng
Feng2, Xiaoying Lin2, Xiaojia Wang3, Xian Wang1, Hongchuan Jin2*
1Department of Medical Oncology, Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China,
2Laboratory of Cancer Epigenetics, Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China,
3Zhejiang Cancer Hospital, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China, 4Cancer Institute, The Second Affiliated Hospital, College of Medicine,
Zhejiang University, Hangzhou, Zhejiang, China
Epidermal growth factor receptor tyrosine kinase inhibitors gefitinib and erlotinib have been widely used in patients with
non-small-cell lung cancer. Unfortunately, the efficacy of EGFR-TKIs is limited because of natural and acquired resistance. As
a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to
play a role in drug resistance of tumor cells. Whether autophagy can be activated by gefitinib or erlotinib and thereby
impair the sensitivity of targeted therapy to lung cancer cells remains unknown. Here, we first report that gefitinib or
erlotinib can induce a high level of autophagy, which was accompanied by the inhibition of the PI3K/Akt/mTOR signaling
pathway. Moreover, cytotoxicity induced by gefitinib or erlotinib was greatly enhanced after autophagy inhibition by the
pharmacological inhibitor chloroquine (CQ) and siRNAs targeting ATG5 and ATG7, the most important components for the
formation of autophagosome. Interestingly, EGFR-TKIs can still induce cell autophagy even after EGFR expression was
reduced by EGFR specific siRNAs. In conclusion, we found that autophagy can be activated by EGFR-TKIs in lung cancer cells
and inhibition of autophagy augmented the growth inhibitory effect of EGFR-TKIs. Autophagy inhibition thus represents a
promising approach to improve the efficacy of EGFR-TKIs in the treatment of patients with advanced non-small-cell lung
Citation: Han W, Pan H, Chen Y, Sun J, Wang Y, et al. (2011) EGFR Tyrosine Kinase Inhibitors Activate Autophagy as a Cytoprotective Response in Human Lung
Cancer Cells. PLoS ONE 6(6): e18691. doi:10.1371/journal.pone.0018691
Editor: Lin Zhang, University of Pennsylvania, United States of America
Received November 11, 2010; Accepted March 15, 2011; Published June 2, 2011
Copyright: ? 2011 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (grant number 30901740, www.nsfc.gov.cn), China National Ministry of
Education Grant (grant number 20090101120124, www.moe.edu.cn), Zhejiang Natural Sciences Foundation Grant (grant number Y2090166, www.zjnsf.net:81),
and Research Projects of Zhejiang Education Department (grant number Y200805751, www.zjedu.gov.cn) to W. Han, and the Fundamental Research Funds for the
Central Universities to H. Jin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (HP); firstname.lastname@example.org (HJ)
Lung cancer is the leading cause of cancer deaths worldwide
. Chemotherapy is still not effective enough for patients with
advanced non–small-cell lung cancer (NSCLC) and the response
rate is only 20% to 35% with a median survival of 10 to 12 months
[2,3]. By targeting molecules critical to cancer development,
targeted therapy alone or in combination with other treatments
was recently recognized as a promising strategy to conquer cancers
including NSCLC .
As one of receptor tyrosine kinases (RTKs) important to cancer
cell growth, proliferation, invasion, and metastasis, epidermal
growth factor receptor (EGFR) was frequently deregulated in
NSCLCs . EGFR over-expression was observed in about 62%
of squamous cell and adenocarcinoma subtypes . EGF can
induce the activation of three signaling pathways important to the
initiation and progression of cancers, Ras/MAPK, PI3K/Akt, and
JAK/STATs . As a result, EGFR became one of the molecules
for the development of targeted therapy to NSCLC. By inhibiting
the tyrosine kinase activity of EGFR, two tyrosine kinase inhibitors
(TKIs) named gefitinib (Iressa, AstraZeneca) and erlotinib
(Tarceva, Genentech) have been developed for the treatment of
NSCLC. Gefitinib and erlotinib can inhibit tumor growth both in
vitro and in vivo. Clinically, both EGFR-TKIs showed good
tolerability and antitumor activity in NSCLC patients with disease
progressing after first line platinum-based chemotherapy [5,8,9].
However, the efficacy of EGFR-TKIs is significantly decreased by
natural and acquired resistance. The mechanism is still largely
unknown although EGFR mutations have been proposed to be
one of mechanisms to influence the sensitivity of EGFR to these
Macroautophagy (hereafter referred to as autophagy) is a self-
proteolysis process in eukaryotic cells that results in the breakdown
of intracellular material within macroautophagosome or lysosomes
[12,13]. Under cellular stress conditions such as nutrient-deficient
environment, autophagy is rapidly activated to provide an
alternative source of energy and thus enable cells to survive
. Autophagy was upregulated during the later stage of tumor
growth because induction of autophagy allows tumor cells to
survive in microenvironments lack of nutrient and oxygen .
Through promoting the survival of tumor cells under unfavorable
conditions, autophagy was proposed as an alternative mechanism
of drug resistance. For example, autophagy contributes to the
resistance of breast cancer cells to bortezomib treatment .
PLoS ONE | www.plosone.org1June 2011 | Volume 6 | Issue 6 | e18691
Inhibition of autophagy could sensitize tumor cells to many
cytotoxic drugs or reverse the resistance to chemotherapeutic
drugs, representing a promising strategy to improve the efficacy of
cancer treatment .
Signaling pathways downstream of EGFR and other RTKs
such as PI3K/Akt pathway are involved in the regulation of
autophagy, indicating a potential link between RTK inhibition
and autophagy. Another TKI named as imatinib indeed can
activate autophagy in respective of cell types [18,19]. In addition,
blockade of macroautophagosome formation enhances the efficacy
of anti-HER2 monoclonal antibody trastuzumab (Tzb) .
However, whether autophagy is associated with gefitinib and
erlotinib treatment in lung cancer cells remains unknown. In the
current study, we first demonstrate that gefitinib or erlotinib
activated autophagy in lung cancer cells and blockage of
autophagy enhanced the effect of gefitinib or erlotinib.
Materials and Methods
Reagents and antibodies
The chemicals used were gefitinib (J&K chemical Ltd.,
G304000), erlotinib (J&K chemical Ltd., E625000) and chloro-
quine (CQ) (J&K chemical Ltd., 147236). The Primary antibodies
were antibodies against microtubule-associated protein 1 light
chain 3 (LC3) (Cell Signaling Technology, #2775), ATG5 (Cell
Signaling Technology, #2630), ATG7 (Cell Signaling Technolo-
gy, #2631), phospho-mTOR (S2448) (Cell Signaling Technology,
#2971), total mTOR (Cell Signaling Technology, #2983),
phospho-P70S6K (T389) (Cell Signaling Technology, #9234),
phospho-AKT (S473) (Cell Signaling Technology, #4051), total
AKT (Cell Signaling Technology, #9272), GAPDH (Cell
Signaling Technology, #3683). The secondary antibodies were
HRP conjugated anti-rabbit (Santa Cruz Biotechnology, sc-2357)
and anti-mouse IgG (Santa Cruz Biotechnology, sc-2371).
The lung cancer cell lines (A549, NCI-H1299, NCI-H292,
NCI-H1650 and SK-MES-1) were bought from cell bank (Chinese
Academy of Sciences). Monolayer culture of cancer cells was
maintained in RPMI 1640 supplemented with 10% (v/v) FBS, and
antibiotics. Stock solution of gefitinib or erlotinib was prepared in
dimethyl-sulphoxide (DMSO) (Sigma, D4540), and diluted with
medium before use. Final concentration of DMSO was ,0.1%.
The cytotoxicity of chemicals against lung cancer cell lines was
determined by MTT assay. Cells were seeded into 96-well plates
(cultured overnight for adherent cells) and treated with chemicals
with different concentrations. After 48-h incubation, 20 ml MTT
(5 mg/ml) was added into each well for 4-h incubation. After that,
the supernatant was removed and 150 ml DMSO was added into
each well in order to solubilize the blue-purple crystals of
formazan. The absorbance was then measured using a model
ELX800 Micro Plate Reader (Bio-Tek Instruments, Inc.) at
Immunofluorescent confocal laser microscopy For LC3
and lysosome co-location
Lysosome was firstly labeled by incubation with Lyso Tracker
(Invitrogen, L7528), a lysosome reporter dye, for 90 min at 37uC.
Cells were collected, fixed and permeabilized with 1% CHAPS
buffer (150 mM NaCl, 10 mM HEPES, 1.0% CHAPS) at room
temperature for 10 min, incubated with anti-LC3 for 2 h at room
temperature, and washed with PBS, incubated for another 45 min
with FITC-conjugated goat anti-rabbit IgG (Beyotime, A0562).
Then, cell nuclei were stained by DAPI (Sigma, D9564). Samples
were examined under a Zeiss LSM 710 confocal microscope
system (Carl Zeiss, Germany). Image was processed with ZEN LE
Treated cells were washed and fixed for 30 min in 2.5%
glutaraldehyde. The samples were treated with 1.5% osmium
tetroxide, dehydrated with acetone and embedded in Durcupan
resin. Thin sections were poststained with lead citrate and
examined in the TECNAI 10 electron microscope (Philips,
Holland) at 60 kV.
Western blot analysis
Western blotting was carried out as previously reported .
The protein was applied to a proper concentration of SDS-
polyacrylamide gel, transferred to a PVDF membrane, and then
detected by the proper primary and secondary antibodies before
visualization with a chemiluminescence kit. Visualization was done
with Image Quant LAS-4000 (Fujifilm, Tokyo, Japan) using image
Multi-Gauge Software (Fujifilm, Tokyo, Japan).
Cells were transfected with either nonspecific siRNA (Qiagen,
1027280), ATG5 siRNA (Qiagen, SI02655310), ATG7 siRNA
(Qiagen, SI02655373) or EGFR siRNA (Cell Signaling Technol-
ogy, #6481) (siRNA final concentration, 100 nmol/L) via
LipofectAMINE RNAi max (Invitrogen, 13778150) according to
the manufacturer’s instructions. Cells were then incubated for
48 h prior to Western blot or MTT assay.
Total RNA was isolated using the Trizol method and cDNA
was synthesized from mRNA with the PrimeScipt MMLV RT
reagent Kit (TakaRa, #6110). Real-time quantitative PCR
reaction was carried out using SYBR green as the fluorescent
reporter (Bio-Rad, 170-8882). QPCR experiments were done on
an ABI-7500 and GAPDH was served as an endogenous control.
Each sample was normalized on the basis of its GAPDH content.
A total of 40 cycles (5-s denaturation at 95uC, 34-s annealingt/
extension at 55uC) were run for each primer. Primer sequences
were as follows: for ATG5, TTC AAT CAG GTT TGG TGG
AGG C (sense) and ATG GCA GTG GAG GAA AGC AGA G
(antisense); for ATG7, ATG CCT GGG CAT CCA GTG AAC
TTC (sense) and CAT CAT TGC AGA AGT AGC AGC CA
(antisense); for GAPDH, GGA GTC AAC GGA TTT GGT
(sense) and GTG ATG GGA TTT CCA TTG AT (antisense).
Unless otherwise stated, data were expressed as the mean 6 SD,
and analyzed by Student’s t test.
EGFR-TKIs induce autophagy in lung cancer cells
To evaluate the activation of autophagy by EGFR-TKIs, the
conversion of LC3-I into LC3-II before and after TKIs treatment
was determined by western blotting analysis. In A549 and NCI-
H1299 cells, both gefitinib and erlotinib can induce the switch of
LC3-I to LC3-II in the dose and time dependent manner,
indicating that autophagy might be activated by both TKIs
(Figure 1A and B). To further confirm it, the compartmentali-
Activation of Autophagy by EGFR Inhibitors
PLoS ONE | www.plosone.org2 June 2011 | Volume 6 | Issue 6 | e18691
zation of LC3 in cells before and after TKIs treatment was
monitored by indirect immunofluorescence staining. Indeed,
TKIs induced the colocalization of LC3-II with lysosome,
demonstrating the formation of autolysosome (Figure 1C and
Figure S1). Ultrastructural analysis by electron microscopy
further confirmed that numerous large autophagic vacuoles with
typical double-layer membrane containing organelle remnants
presented in gefitinib-treated A549 cells rather than untreated
cells (Figure 2A, B and C). Similar results were obtained with
erlotinib (Figure 2D and E).
We next examined the effects of EGFR-TKIs on the expression
of ATG5 and ATG7, two critical components in regulating the
formation of autophagosomes . Both EGFR-TKIs increased
ATG5 and ATG7 at the mRNA or protein levels (Figure 3),
confirming the induction of autophagy by EGFR-TKIs.
Inhibition of Akt/mTOR/p70S6K signaling pathway by
Previous studies have shown that PI3K/Akt/mTOR/p70S6K
axis plays an important role in the inhibition of autophagy [23,24],
Figure 1. EGFR-TKIs dose- and time-dependently induced the formation of LC3-II, and formation of autolysosomes in lung cancer
cells. A and B, lung cancer cells were incubated with varying concentrations of gefitinib or erlotinib for 24 hours or incubated with 25 mM gefitinib
or erlotinib for appropriate intervals. The switch of LC3-I to LC3-II was detected by immunoblotting. C, A549 or NCI-H1299 cells were treated with
DMSO or 25 mM gefitinib for 24 h. Cells were labeled with fluorescence and imaged by confocal microscope. Red, lyso tracker-labeled lysosomes;
Green, FITC-labeled LC3; Blue, DAPI-labeled nucleus. The overlay was shown in the right column. The orange-stained cells indicated LC3 co-located
Activation of Autophagy by EGFR Inhibitors
PLoS ONE | www.plosone.org3 June 2011 | Volume 6 | Issue 6 | e18691
we therefore investigate the effect of gefitinib and erlotinib on this
autophagy-suppressive signaling pathway. Indeed, phosphoryla-
tion of AKT, mTOR and p70S6K in both A549 and H1299 cells
were significantly reduced by gefitinib and erlotinib in a time-
dependent manner (Figure 4A and B). However, no changes of
PTEN expression were detected after TKIs treatment (Figure 4C
Blockage of autophagy enhance EGFR-TKIs-induced cell
Many studies have demonstrated that autophagy may serve as a
protective response preventing tumor cells from therapy-induced
cell death [17,20,25,26,27,28,29,30]. In lung cancer cell line NCI-
H292 and NCI-H1650 that are relatively sensitive to gefitinib and
erlotinib, no autophagy was detected after gefitinib or erlotinib
treatment (Figure 5A). However, autophagy was detected in
EGFR-TKIs treated cancer cells that are relatively resistant to
EGFR-TKIs, such as A549, NCI-H1299 and SK-MES-1 cells
(Figure 1 and 5A). These data indicated that autophagy may
impair the sensitivity of cancer cells to EGFR-TKIs. To test this
consumption, we compared the growth inhibitory effect of
gefitinib or erlotinib on cancer cells before and after pharmaco-
logical and genetic inhibition of autophagy. As shown in Figure 5B,
CQ which can impair the function of lysosomes and inhibit
autophagy at late stage significantly augmented growth inhibition
induced by gefitinib or erlotinib in A549, NCI-H1299 and SK-
MES-1 that are relatively resistant to EGFR-TKIs. However, it
could not inhibit lung cancer cell growth by its own. (Figure 5B).
Similarly, growth inhibition induced by gefitinib or erlotinib in
A549 cells was enhanced after autophagy was inhibited by the
knockdown of ATG5 or ATG7 (Figure 5C and D), two essential
components for the formation of autophagosome, confirming that
Figure 2. TEM depicting ultrastructures of autophagosome in A549 cells treated EGFR-TKIs. A549 cells were treated with 25 mM gefitinib
for 24 h (A, DMSO; B, Gefitinb, low power; C, Gefitinb, high power). D (low resolution) and E (high resolution), AGS cells were treated with 25 mM
erlotinib for 24 h (D, erlotinib, low power; E, erlotinib, high power). Numerous autophagical vacuoles with typical double-layer membrane containing
organelle remnants were highlighted by arrows. Bar=1 mm.
Figure 3. Effects of EGFR-TKIs on the expression of ATG5 and
ATG7. A, A549 cells were treated with 25 mM gefitinib for 6 h and the
mRNA level of ATG5/7 was measured by Real time RT-PCR as described
in M&M. RQ, relative quantity. black, gefitinib-treated cells; gray, control
cells. B, Immunoblotting for Atg5 or Atg7 using lysates from A549
treated with varying concentrations of gefitinib or erlotinib for 24 hr.
Data were expressed as the mean 6 SD, and analyzed by Student’s
t test. ** P,0.01.
Activation of Autophagy by EGFR Inhibitors
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autophagy protected tumor cells from EGFR-TKIs induced cell
EGFR-TKIs induce autophagy in cancer cells with EGFR-
Next, we would like to know the role of EGFR in the gefitinib or
erlotinib induced autophagy. Two specific siRNAs were used to
knockdown EGFR expression. SiRNA 1 could reduce EGFR
expression greatly while siRNA 2 which had moderate effect on
EGFR expression (Figure 6A and B). Interestingly, autophagy
activated by either gefitinib or erlotinib was not inhibited but
augmented after EGFR expression was dramatically reduced by
siRNA 1 (Figure 6C and D). In contrast, siRNA 2 hardly had any
influence on autophagy induced by both EGFR-TKIs (Figure 6C
Autophagy was designated as programmed cell death type II,
whereas apoptosis is well-known as programmed cell death type I.
However, recently autophagy was found to promote cellular
survival under unfavorable conditions such as deprivation of
amino acids or ATPs, revealing a new role of autophagy in cancer
Autophagy is morphologically characterized by the appearance
of ‘‘double-membrane’’ vacuoles (autophagosomes) in the cyto-
plasm. In addition, the mammalian homologue of the yeast
protein Apg8p, (also named LC3), is found to be a specific
biochemical marker for autophagy. Newly synthesized LC3
termed LC3-1 is evenly distributed throughout the cytoplasm.
Upon induction of autophagy, some LC3-I is converted into LC3-
II, which is tightly bound to the autophagosomal membranes
forming ring-shaped structures in the cytosol. We confirmed
biochemically and morphologically that autophagy can be
activated by gefitinib or erlotinib, two well-used EGFR-TKIs, in
two independent lung cancer cell lines. Interestingly, a very recent
paper reported that autophagy can be induced by anti-EGFR
antibodies . Both EGFR antibodies and EGFR-TKIs are
widely used to treat cancer patient by inhibition of EGFR,
revealing a new link between EGFR inhibition and autophagy
Autophagy can be activated as the cellular response to cancer
therapy. A number of cancer therapeutics including DNA-
damaging chemotherapeutics, endocrine therapies (e.g. tamoxifen)
and radiation therapy have been found to induce autophagy in
vitro and in vivo [32,33,34]. Recently, it was found that autophagy
can be activated and protected tumor cells from targeted
therapies, such as the imatinib mesylate in philadelphia chromo-
some–positive cells , trastuzumab in breast cancer , Src
family kinase inhibitors in prostate cancer , proteasome
inhibitors in prostate cancer . Consistently, we found that
autophagy can be activated by gefitinib and erlotinib in lung
cancer and promote cellular survival in the target therapy using
EGFR-TKIs. Blockage of autophagy by pharmacological or
genetic approaches greatly enhanced the growth inhibitory effect
of gefitinib or erlotinib. Thus, inhibition of autophagy has the
potential to improve the clinical efficacy of EGFR-TKIs for cancer
As a sensor of amino acids and ATP, mTOR negatively
regulates autophagy. Indeed, we found that both TKIs can inhibit
the activation of mTOR as well its upstream regulator, PI3K/Akt.
Another signaling pathway important to autophagy is Raf/MAPK
pathway . However, we failed to find a clear correlation
between Raf/MAPK pathway and autophagy in cell lines we used.
This is probably due to oncogenic mutations predominantly
occurred in Ras/MAPK pathways. For instance, k-Ras gene was
known to be mutated in A549 cells. Clinically, patients with k-Ras
mutations failed to response to TKIs treatment. It would be
interesting to know whether patients with k-Ras mutations could
benefit from the combination of TKIs and inhibitors of autophagy.
Figure 4. EGFR-TKIs induced autophagy by inhibiting the Akt/mTOR/p70S6K signaling pathway in A549 and H1299 cells. The
phosphorylation of mTOR, Akt and p70s6k in A549 (A) or NCI-H1299 (B) cells treated with gefitinib or erlotinib were determined by western blotting.
The expression of PTEN in A549 (C) or NCI-H1299 (D) cells treated with varying concentrations of gefitinib or erlotinib were detected by western
Activation of Autophagy by EGFR Inhibitors
PLoS ONE | www.plosone.org5 June 2011 | Volume 6 | Issue 6 | e18691
Interestingly, our results indicated that gefitinib or erlotinib
induced autophagy might be EGFR independent. As shown in
Figure 6, both EGFR-TKIs could induce autophagy even EGFR
expression was greatly reduced. Although gefitinib and erlotinib
were developed as the specific inhibitors targeted to the kinase
domain of EGFR, however, recent results indicted that these two
inhibitors can have other targets, such as non-receptor tyrosine
kinases that acts also upstream of PI3K/Akt/mTOR pathway
. Consistently, large scale clinical trials confirmed that
measurement of EGFR expression by immunohistochemistry
was not useful for picking up patients to be benefited from
gefitinib therapy. Certainly, we still could not completely exclude
the relevance of EGFR in gefitinib or erlotinib induced autophagy
since certain amount of EGFR was still detected after knockdown
by EGFR siRNAs. Indeed, EGFR-TKIs activated autophagy was
much more enhanced by siRNA 1 which displayed better
knockdown efficiency than siRNA 2 (Figure 6B). Hence, we need
further investigations to clarify the role of EGFR in the gefitinib or
erlotinib-induced autophagy. Nevertheless, EGFR-TKIs and
EGFR siRNA had a synergetic effect on the induction of
autophagy, which could be specifically inhibited to increase the
clinical efficacy of targeted therapy.
In summary, our work reinforced the notion that cancer cells
can survive in a stressful environment, following inhibition of
critical oncogenic pathways, by inducing autophagy. These tumor
cells are primed to resume proliferation once drug concentration
drops after drug withdrawal due to toxicity or mutations develop
to confer drug resistance. Thus, in combination of therapeutic
strategies that aim to inhibit autophagy in patients treated with
conventional chemotherapy or novel targeted therapy with
EGFR-TKIs represents a promising approach with higher efficacy
for cancer patients.
Figure 5. Inhibition of autophagy enhances the cytotoxity of EGFR-TKIs in lung cancer cells. A, Autophagy was induced by EGFR-TKIs in
resistant but not sensitive lung cancer cells. LC3 expression in lung cancer cells incubated with 25 mM gefitinib or erlotinib for 24 h were analyzed by
immunoblotting. B, The viability of cancer cells treated with 12.5 mM gefitinib or erlotinib for 48 h with or without 5 mM CQ were measured by MTT
assay. C, The expression of ATG5 and ATG7 in A549 cells transiently transfected with negative control siRNA, Atg7 or Atg5 siRNA were determined by
immunoblotting. D, Cells transiently transfected with negative control siRNA, Atg7 or Atg5 siRNA were treated with 12.5 mM gefitinib or 25 mM
erlotinib for 48 h. The effect of EGFR-TKIs on cancer cells with or without ATG5 or ATG7 knockdown were analyzed by MTT assay. NC, Control siRNA.
Data were expressed as the mean 6 SD, and analyzed by Student’s t test. * P,0.05, ** P,0.01.
Activation of Autophagy by EGFR Inhibitors
PLoS ONE | www.plosone.org6 June 2011 | Volume 6 | Issue 6 | e18691
cells treated by gefitinib. The percentage of cells with orange
signals was calculated by counting cells in 3–4 fields under
microscope. The data were represented as the mean 6 SD.
Quantification of autophagy in lung cancer
Conceived and designed the experiments: WH XW HJ. Performed the
experiments: WH YC JS YW JL WG LF XL. Analyzed the data: WH HP
XW XJW HJ. Wrote the paper: WH XW HJ.
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Figure 6. EGFR-TKIs induced autophagy in EGFR-knockdown cells. EGFR expression in A549 (A) or H1299 (B) cells transfected with control
siRNA, EGFR siRNA1 or siRNA2 were determined by western blotting. LC3 expression in EGFR-TKIs treated A549 (C) or H1299 (D) cells with or without
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Activation of Autophagy by EGFR Inhibitors
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