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Supplementary Information

Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and

migration by promoting EGFR endocytic degradation

Meng-Yen Li, Pei-Lun Lai, Yu-Ting Chou, Ai-Pei Chi, Yu-Zhen Mi, Kay-Hooi Khoo,

Geen-Dong Chang, Cheng-Wen Wu, Tzu-Ching Meng and Guang-Chao Chen *

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Supplementary Methods

Drosophila strains and genetics

Flies were raised at 25°C following standard procedures unless otherwise noted. The following

Drosophila strains were used: en-Gal4, Slbo-Gal4, dPtpmeg1, UAS-RasV12, UAS-Dp110CAAX,

and RasGAP1B2 were obtained from the Bloomington Stock Center. UAS-myrAkt1 and EgfrElp2

were gifts from Henry Sun (Academia Sinica). The UAS-dPtpmeg transgene was generated by

subcloning the dPtpmeg from LD13416 into the pUAST vector. The UAS-dPtpmeg-CS mutant,

in which the catalytic site cysteine residue 781 was changed to a serine, was generated by PCR

mutagenesis and was subcloned into the pUAST vector. The UAS-Eps15 transgene was generated

by subcloning the Eps15 from SD09478 into the pUAST vector. The dPtpmeg overexpression

clones were generated using the Flip-out technique 3.

In vitro substrate-trapping and MS-based analysis

HA-tagged PTP domain of dPtpmeg (dPtpmeg-PTP-WT, amino acids 497 to 856) or the PTP

domain substrate-trapping mutant (dPtpmeg-PTP-DA, D691A) were generated by PCR and

subcloned into pROEX-HTa. In vitro substrate-trapping and MS-based analysis was performed as

previously described 4.

In vitro kinase assay

FLAG-tagged full-length Eps15 WT or YF were in vitro translated and in vitro phosphorylated by

EGFR. In vitro translation was performed using the TNT quick coupled transcription/translation

system (Promega) according to manufacturer's instructions. In vitro kinase assay was performed

as previously described5.

In vitro wound-healing and transwell migration assay

H1975 lung cancer cells expressing PTPN3, Eps15-Y850F, or an empty vector control were

plated onto six-well culture plates in RPMI containing 10% FBS. After 24 h, the cell monolayers

were wounded manually by scratching with a pipette tip, and rinsed with PBS. Fresh RPMI with

1% FBS and 10 μg/ml mitomycin C was added to follow healing for 24 h. Cells were

photographed at 0 h, 6 h and 24 h after wounding using an Olympus IX70 phase contrast

microscopy and an Olympus DP30BW camera. For transwell migration assays, 1 × 105 cells were

seeded into and grown in low serum medium (1% FBS) on the top chambers of transwells (8 μm

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pores, Millipore). The lower chamber was filled with medium containing 10% FBS. Mitomycin C

(Sigma) was added to inhibit cell proliferation. After 24 h, the migrated cells on the lower surface

of membrane were fixed and stained with 0.5% Crystal Violet (Sigma). The absorbance was

measured at 570 nm using a microplate reader (Tecan).

Immunofluorescence

Ovaries and wing imaginal discs were fixed with 4% paraformaldehyde in PBS for 20 min at

room temperature. Tissues were permeabilized with 0.3% Triton/PBS and blocked with 5%

normal goat serum in PBST (PBS + 0.1% Triton). Samples were then incubated with primary

antibodies overnight at 4°C. On the following day, samples were washed with PBST and

incubated with fluorescent-labeled secondary antibodies for 2 h at room temperature. For

immunofluorescent analysis of mammalian cells, cells grown on coverslips were fixed with 4%

paraformaldehyde for 15 min and permeabilized with 0.1% Triton/PBS for 5 min. They were then

incubated with primary antibodies overnight at 4°C, followed by incubation with secondary

antibodies. Nucleus was stained using DAPI (1 μg/ml). Samples were visualized under a confocal

laser scanning microscope (Zeiss LSM510).

Cell growth and colony formation assay

Cell growth assay was carried out using the WST-1 kit (Roche) according to the manufacturer's

instructions. Briefly, lentiviral-infected H1975 cells and CL1-5 cells (1 × 103cells/well) were

seeded on 96-well plates and maintained in RPMI-1640 medium. WST-1 reagent was added to

the cells and incubated for 1 hr at 37°C. The absorbance at 450 nm was measured using a

microplate reader (Tecan). For colony formation assay, virus-infected H1975 and CL1-5 cells

(500 cells/well) were seeded in a 6-well plate. Colonies were fixed and stained with 4%

formaldehyde/0.2% crystal violet and counted 10 days after plating.

Immunohistochemistry

Immunohistochemical detection of EGFR and phospho-Eps15 was performed on 5-µm-thick

paraffin sections. The sections were deparaffinized and subjected to antigen retrieval with 10

mM sodium citrate buffer (pH 6.0) at 100°C. Endogenous peroxidase activity and nonspecific

binding were blocked by sequential incubation in TBS-T with 3% hydrogen peroxide for 5 min

and 5% goat serum for 1 hr. Sections were then incubated with EGFR or phsopho-Eps15

antibody at 4°C for 16 hr, and then with HRP-conjugated secondary antibody. The slides were

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stained with diaminobenzidine hydrochloride (DAB), washed, and counterstained with

hematoxylin. Negative controls were prepared by substitution of rabbit IgG for the primary

antibody

Flow cytometry analysis

For internalization studies, cells were incubated with 100 ng/ml EGF-Alexa Fluor 488

(Invitrogen) for 1 hr at 4°C and the cells were washed with ice-cold PBS. We then allowed

endocytosis to occur at 37°C for various intervals (0, 5, 10 and 15 min). Cells were subsequently

washed with acid-stripping buffer (50 mM glycine, 150 mM NaCl, pH 3.0), trypsinized, and

fixed in BD Cytofix/Cytoperm solution (BD Bioscience) for 15 min at 4°C. Flow cytometry, data

collection, and analysis were performed using BD FACSalibur (BD Bioscience).

References

1. Stocker H, Andjelkovic M, Oldham S, Laffargue M, Wymann MP, Hemmings BA, et al.

Living with lethal PIP3 levels: viability of flies lacking PTEN restored by a PH domain

mutation in Akt/PKB. Science 2002, 295(5562): 2088-2091.

2. Baker NE, Rubin GM. Ellipse mutations in the Drosophila homologue of the EGF

receptor affect pattern formation, cell division, and cell death in eye imaginal discs.

Developmental biology 1992, 150(2): 381-396.

3. Struhl G, Basler K. Organizing activity of wingless protein in Drosophila. Cell 1993,

72(4): 527-540.

4. Ku HY, Wu CL, Rabinow L, Chen GC, Meng TC. Organization of F-actin via concerted

regulation of Kette by PTP61F and dAbl. Mol Cell Biol 2009, 29(13): 3623-3632.

5. Chen GC, Lee JY, Tang HW, Debnath J, Thomas SM, Settleman J. Genetic interactions

between Drosophila melanogaster Atg1 and paxillin reveal a role for paxillin in

autophagosome formation. Autophagy 2008, 4(1): 37-45.

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Supplementary Figure

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Supplementary Figure S1. PTPN3 does not interact with EGFR. (A) Ectopic expression of

Eps15 with en-Gal4 caused a wing notch phenotype. Eps15-induced wing defects could be

suppressed by coexpression of dPtpmeg. (B) HEK293T cells transfected with Myc-tagged

full-length Eps15, together with FLAG-tagged PTPN23-WT, PTPN23-CS or an empty vector

control were stimulated with EGF (100 ng/ml) for 5 min and cell lysates were

immunoprecipitated with anti-Myc antibody. The immunoprecipitates and total cell lysates (TCL)

were analyzed by immunoblotting with antibodies as indicated. (F) HEK293T cells transfected

with Myc-tagged full-length Eps15, together with FLAG-tagged Shp2-WT, Shp2-CS or an empty

vector control were stimulated with EGF (100 ng/ml) for 5 min and cell lysates were

immunoprecipitated with anti-Myc antibody. The immunoprecipitates and total cell lysates were

analyzed by immunoblotting with antibodies as indicated. (D) Schematic presentation of the

domain structure and deletion mutants of Eps15 and PTPN3. (E) FLAG-tagged full-length Eps15

WT or YF were in vitro translated and in vitro phosphorylated by EGFR. Phosphorylation of

FLAG-Eps15 WT or YF was analyzed by immunoblotting with pEps15 (Y850) antibody. (F)

Equal amounts of purified GST, GST-PTPN3ΔN WT or DA fusion proteins were mixed with

FLAG-tagged Eps15 WT or YF prepared from (E) for in vitro pull-down assays. (G) HEK293T

cells transfected with HA-tagged PTPN3-WT, PTPN3-DA, or an empty vector control were

stimulated with EGF (100 ng/ml) for 5 min and cell lysates were immunoprecipitated with

anti-HA antibody. The immunoprecipitates and total cell lysates (TCL) were analyzed by

immunoblotting with antibodies as indicated. (H) HEK293T cells transfected with HA-tagged

PTPN3-WT, PTPN3-CS, or an empty vector control were stimulated with EGF (100 ng/ml) for 5

min and cell lysates were immunoprecipitated with anti-EGFR antibody. The immunoprecipitates

and TCL were analyzed by immunoblotting with antibodies as indicated.

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Supplementary Figure S2. PTPN3 downregulates EGFR signaling in A549 cells. (A) A549

cells were treated without or with EGF (100 ng/ml) for 5 min and immunostained with

anti-PTPN3 (red) and anti-EGFR (green) antibodies. Nuclei were stained with DAPI in blue. The

insets show a higher magnification of the area enclosed within the white box. Bar, 10 µm. (B)

A549 cells stably expressing HA-tagged PTPN3 or an empty vector control were incubated with

100 ng/ml EGF for the indicated times. Cell lysates were analyzed by immunoblotting with

antibodies as indicated.

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Supplementary Figure S3. PTPN3-mediated Eps15 dephosphorylation does not affect EGF

internalization. (A-B) H1975 cells stably expressing HA-tagged PTPN3, FLAG-Eps15-Y850F,

or control were stimulated with EGF-488 (100 ng/ml) for 5 min and stained with anti-EEA1 (A)

or anti-Rab11 (B) antibody. Nuclei were stained with DAPI in blue. The insets show a higher

magnification of the area enclosed within the white box. Bar, 10 µm. (C) PTPN3 induces EGFR

degradation in a phosphatase activity-dependent manner. H1975 cells stably expressing

HA-tagged PTPN3, PTPN3-CS, or an empty vector control were serum starved overnight and

treated with EGF (100 ng/ml) for the indicated times (0, 5, 15, and 30 min). Cell lysates were

analyzed by immunoblotting with antibodies as indicated.

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Supplementary Figure S4. Ectopic expression of PTPN3 and Eps15-Y850F reduce EGFR

but not Met levels. H1975 cells stably expressing HA-tagged PTPN3, FLAG-tagged

Eps15-Y850F, or an empty vector control were were stimulated with 50 ng/ml HGF for the

indicated times (0, 5, 15, 30 min). Cell lysates were analyzed by immunoblotting with antibodies

as indicated.

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Supplementary Figure S5. PTPN3 and Eps15-Y850F promote proteasome-independent

EGFR degradation. H1975 cells stably expressing HA-tagged PTPN3 (A), FLAG-tagged

Eps15-Y850F (B), or an empty vector control were treated with 50 μM MG132 for 2 hr, followed

by incubation with 100 ng/ml EGF for the indicated times (0, 5, 15, 30 min). Cell lysates were

analyzed by immunoblotting with antibodies as indicated.

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Supplementary Figure S6. Colocalization of EGF-488 and caveolin-1 in PTPN3 and

Eps15-Y850F expressing cells. (A-B) H1975 cells stably expressing an empty vector control,

HA-tagged PTPN3, or FLAG-tagged Eps15-Y850F were treated with EGF-Alexa 488 (100 ng/ml)

for 5 min and stained with anti-clathrin (A) or anti-caveolin-1 (B) antibody. Nuclei were stained

with DAPI in blue. The insets show a higher magnification of the area enclosed within the white

box. Bar, 10 µm. (C) Percentage colocalization of EGF-Alexa 488 with clathrin in (A). (D)

Percentage colocalization of EGF-Alexa 488 with caveolin-1 in (B). Data in (C) and (D) are

represented as mean ± SD of triplicates, with an average of ten cells scored per experiment. ***,

P < 0.001.

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Supplementary Figure S7. Ectopic expression of PTPN3 and Eps15-Y850F reduce EGFR

but suppression by filipin. (A-B) H1975 cells stably expressing HA-tagged PTPN3 (A),

FLAG-tagged Eps15-Y850F (B), or an empty vector control were treated with 10 μg/ml filipin

for 60 min, followed by incubation with 100 ng/ml EGF for the indicated times (0, 5, 15, 30 min).

Cell lysates were analyzed by immunoblotting with antibodies as indicated.

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Supplementary Figure S8. Depletion of PTPN3 does not affect clathrin-mediated

endocytosis of EGFR. (A) Relative expression levels of PTPN3 in six NSCLC cell lines (H520,

H1975, CL1-5, A549, H1299, and H928) were measured by quantitative real-time PCR

(qRT-PCR). Data are expressed as a fold change compared with the normal human lung cells

(BEAS-2B). Data represent the mean ± SD of three independent experiments (B) Lysates of

CL1-5 cells infected with scramble control, shPTPN3-A1, or shPTPN3-D1 were immunoblotted

with anti-PTPN3 and anti-tubulin antibodies. (C-D) Lysates of CL1-5 cells infected with

scramble control, shPTPN3-A1 were fractionated by sucrose density gradient centrifugation, and

aliquots were immunoblotted with anti-EGFR, anti-clathrin, and anti-caveolin-1 antibodies.

Fractions 3-5 were enriched with caveolin-1 and denoted as lipid raft fractions, whereas fractions

8-10 were enriched with clathrin and denoted as non-lipid raft

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Supplementary Table S1. The table represents percentages of internalized EGF measured by

flow cytometry of three independent experiments.