TUFM downregulation induces epithelial-mesenchymal transition
and invasion in lung cancer cells via a mechanism involving
AMPK-GSK3β signaling
Journal name: Cellular and Molecular Life Sciences
Kai He1,2, Xiaojie Guo1,2, Yi Liu1, Jingsong Li1, Ying Hu1, Dongmei Wang1, and
Jianguo Song1
1. State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320
Yue-Yang Road, Shanghai 200031, China
2. These authors contributed equally to this article.
Corresponding Author: Jianguo Song, State Key Laboratory of Cell Biology,
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031,
China. Phone/Fax: +86 21 54921167; E-mail: jgsong@sibcb.ac.cn
Supplementary Figure S1 TUFM levels are negatively correlated with EMT in
breast cancer cells. (A) The cell morphology of MCF7, MDA-MB-453 and
MDA-MB-231 cells. Cells were photographed at 100 × magnification. MCF7 cells
exhibited
a
compact
epithelial
morphology,
whereas
MDA-MB-453
and
MDA-MB-231 cells displayed scattered and spindle-like mesenchymal shapes. (B)
The cell migration of the above cells was examined via transwell assay. The data
shown are a representative result of three independent experiments and the statistical
analysis. (C) The cellular levels of ZO-1, E-cadherin, vimentin and TUFM of MCF7,
MDA-MB-453, and MDA-MB-231 cells were examined by immunoblotting.
Supplementary Figure S2 TUFM expression levels in various bladder cancers.
Oncomine box plots of TUFM expression levels in infiltrating bladder cancer
urothelial carcinoma and superficial bladder cancer. The numbers of samples and P
values are shown.
Supplementary Figure S3. Overexpression of TUFM in different lung cancer
cells. (A) The morphology of A549 cells was photographed at 100 × magnification.
(B) The TUFM protein levels in control and mouse TUFM overexpressed A549 cells
were determined via immunoblotting. (C) The protein levels of E-cadherin and
N-cadherin in indicated A549 cells were determined via immunoblotting. (D) The
morphology of GFP or human TUFM overexpressed H1299 and H1650 cells were
photographed at 100 × magnification. (E) The protein levels of E-cadherin,
N-cadherin and vimentin in GFP or human TUFM overexpressed H1299 and H1650
cells were determined via immunoblotting.
Supplementary Figure S4 TUFM knockdown facilitates the survival of
circulating tumor cells. 2 months after tail vein injection, the circulating tumor cells
in blood were determined using real-time PCR. The data shown are the means ± s.d.
of three independent experiments.
Supplementary Figure S5 AMPK activation is required for TGF-1-induced
EMT in A549 cells. (A) A549 cells were treated with 5 ng/ml of TGF-1 for the
indicated time. The expression of AMPK and p-AMPK (T172) was determined via
immunoblotting. (B) A549 cells were treated with TGF-1 (2 ng/ml) and Compound
C (10 M) for 48 h. EMT was examined by observing the cell morphology and by
immunoblotting of E-cadherin. (C) Control and AMPK-knockdown A549 cells were
treated with TGF-1 (2 ng/ml) for 48 h. EMT was examined by assaying the changes
in cell morphology and the levels of E-cadherin and N-cadherin.
Supplementary Figure S6. The effect of AMPK on TGF-1-induced EMT and
Smad phosphorylation in A549 cells. A549 cells were treated with TGF-1 (2
ng/ml), the AMPK activator AICAR (1 mM) or the inhibitor of TGF- receptor
SB431542 (2M) for 48 h. EMT was examined by observing the cell morphology (A)
and by immunoblotting of E-cadherin and N-cadherin (B). Control, AMPK or
CA-AMPKoverexpressed A549 cells were treated with TGF-1 (2 ng/ml) for 48h.
EMT was examined by immunoblotting of E-cadherin and N-cadherin (C) and by
observing the cell morphology (D). (E) Cells were pre-treated with AICAR (1 mM) 3
h before the addition of TGF-1 (2 ng/ml) and SB431542 (2M). The
phosphorylation of Smad2 and Smad3 were detected 1h after TGF-1 (2 ng/ml)
treatment. (F) The phosphorylation of Smad2 and Smad3 were detected 1h after
TGF-1 (2 ng/ml) treatment.
Supplementary Figure S7 TUFM knockdown increases the level of Axin2 mRNA.
(A) The relative levels of Axin2 mRNA in control and TUFM-knockdown A549 cells
were quantified using real-time PCR. (B) The relative levels of Axin2 mRNA in cells
in response to Compound C (10 M, 24 h) were quantified using real-time PCR. The
data shown are the means ± s.d. of three independent experiments.
Supplementary Figure S8 Different effect of TUFM knockdown on -catenin
nuclear
translocation.
(A)
The
nuclear
-catenin
expression
level
in
TUFM-knockdown H446 cells was detected via immunoblotting. (B) The location of
-catenin in control and TUFM-knockdown MCF7 cells was detected via
immunofluorescent staining.
Supplementary Figure S9 Knockdown of -catenin abrogates TUFM knockdown
induces increase of -catenin in nuclei. The protein levels of nuclear -catenin in
indicated cells were detected by immunoblotting. Tubulin and PARP were used as
loading controls for the cytoplasmic and nuclear fractions, respectively.
Supplementary Figure S10 Low-glucose sensitivity of A549 cells. Control and
TUFM-knockdown A549 cells were cultured in high-glucose (25 mM) or low-glucose
(2 mM) medium for two days, and apoptosis was examined via FACS. The data
shown are the means ± s.d. of three independent experiments.
Supplementary Figure S11 Knockdown of TUFM promotes autophagic induction
in A549 cells. The induction of autophagy in A549 cells was detected by examining
the protein level of LC3I and LC3II.