Supplementary Table S1

Proteins associated with nuclear RON identified using co-immunoprecipitation-LC-MS/MS

Accession no. Gene Symbol Protein name

NP_000998.1 RPS4X 40S ribosomal protein S4, X isoform

NP_000997.1 RPS3A 40S ribosomal protein S3a

NP_002939.2 RPL15 60S ribosomal protein L15

NP_000961.2 RPL6 60S ribosomal protein L6

NP_001613.2 AHSG Alpha-2-HS-glycoprotein

NP_001091.1 ACTA Actin

NP_663736.2 ACBD5 Acyl-CoA-binding domain-containing protein 5

NP_055299.1 MOCS3 Adenylyl transferase and sulfur transferase

NP_001613.2 AHSG Alpha-2-HS-glycoprotein

NP_001002858.1 ANXA2 Annexin A2

NP_001460.1 XRCC6 ATP-dependent DNA helicase 2 subunit 1

NP_001193723.1 ARID4B AT-rich interactive domain-containing protein 4B

NP_001264.2 CHD4 Chromodomain-helicase-DNA-binding protein 4 Accession no. Gene Symbol Protein name

NP_004850.1 CLTC Clathrin heavy chain 1

NP_009029.3 CLTCL1 Clathrin heavy chain 2

NP_060282.1 CHCHD3 Coiled-coil-helix-coiled-coil-helix domain-containing protein 3

NP_001129612.1 CMPK1 Cytidylate kinase

NP_060139.2 ERCC6L DNA excision repair protein ERCC-6-like

NP_004517.2 MCM2 DNA replication licensing factor MCM2

NP_008835.5 PRKDC DNA-dependent protein kinase catalytic subunit

NP_005207.2 DDOST Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1

NP_001393.1 EEF1A1 Elongation factor 1-alpha 1

NP_079272.4 GFM1 Elongation factor G

NP_112598.2 EPPK1 Epiplakin

NP_001104026.1 FLNA Filamin-A

NP_002037.2 GAPDH Glyceraldehyde-3-phosphate dehydrogenase

NP_003504.2 H2A1B Histone H2A type 1-B/E

NP_006796.1 HNRNPA0 Heterogeneous nuclear ribonucleoprotein A0 Accession no. Gene Symbol Protein name

NP_001011724.1 HNRNPA1L2 Heterogeneous nuclear ribonucleoprotein A1-like protein 2

NP_919223.1 HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3

NP_112740.1 HNRNPDL Heterogeneous nuclear ribonucleoprotein D-like

NP_004957.1 HNRNPF Heterogeneous nuclear ribonucleoprotein F

NP_112533.1 HNRNPA2B1 Heterogeneous nuclear ribonucleoprotein H3

NP_002131.2 HNRNPK Heterogeneous nuclear ribonucleoprotein K

NP_002127.1 HNRPNA1 Heterogeneous nuclear ribonucleoprotein L

NP_006363.4 SYNCRIP Heterogeneous nuclear ribonucleoprotein Q

NP_001095868.1 HNRNPR Heterogeneous nuclear ribonucleoprotein R

NP_114032.2 HNRNPU Heterogeneous nuclear ribonucleoprotein U

NP_000179.2 HK1 Hexokinase-1

NP_004506.2 ILF2 Interleukin enhancer-binding factor 2

NP_733821.1 LMNA Lamin A/C

NP_001185486.1 LMNB1 Lamin B1

NP_116126.3 LMNB2 Lamin-B2 Accession no. Gene Symbol Protein name

NP_061322.2 MATR3 Matrin-3

NP_078873.2 MAP7D3 MAP7 domain-containing protein 3

NP_060476.2 TRMU Mitochondrial tRNA-specific 2-thiouridylase 1

NP_001093639.1 IMMT Mitochondrial inner membrane protein

NP_005372.2 NCL Nucleolin

NP_000260.1 NM23 Nucleoside diphosphate kinase

NP_002810.1 PTBP1 Polypyrimidine tract-binding protein 1

NP_001077007.1 POTEE POTE ankyrin domain family member E

NP_006377.2 DDX17 Probable ATP-dependent RNA helicase

NP_006182.2 PA2G4 Proliferation-associated protein 2G4

NP_002625.1 PHB Prohibitin

NP_001238978.1 PCMT Protein-L-isoaspartate O-methyltransferase

NP_689481.2 PARS2 Prolyl-tRNA synthetase

NP_006388.2 RNASEH2A Ribonuclease H2 subunit A

NP_001243764.1 QTRTD1 Queuine tRNA-ribosyl transferase Accession no. Gene Symbol Protein name

NP_001258780.1 RPAGC Ras-related GTP-binding protein C

NP_001779.3 SEPT7 Septin-7

NP_005057.1 SFPQ Splicing factor, proline- and glutamine-rich

NP_036565.2 SF3B1 Splicing factor3B subunit 1

NP_006073.2 TUBA1B Tubulin alpha-1B chain

NP_003248.3 TJP1 Tight junction protein ZO-1

NP_004085.1 EIF2S1 Translation initiation factor IF-2

NP_054733.2 SNRNP200 U5 small nuclear ribonucleoprotein 200 kDa helicase

P03993 UBIQ Ubiquitin Supplementary Table S2

The emPAI value corresponds to the relative abundance of the MS/MS identification

Protein Accession Protein Name Protein score emPAI

UBIQ_HUMAN Ubiquitin OS = Homo sapiens GN = RPS27A PE = 1 SV = 1 38 0.48

ROA2_HUMAN Heterogeneous nuclear ribonucleoproteins A2/B1 118 0.33

OS=Homo sapiens GN = HNRNPA2B1 PE = 1 SV = 2

H2A1B_HUMAN Histone H2A type 1-B/E 92 0.28

RA1L2_HUMAN Heterogeneous nuclear ribonucleoprotein A1-like protein 2 64 0.23

RL15_HUMAN 60S ribosomal protein L15 77 0.16

RS4X_HUMAN 40S ribosomal protein S4, X isoform 67 0.13

RL6_HUMAN 60S ribosomal protein L6 104 0.12

ROA0_HUMAN Heterogeneous nuclear ribonucleoprotein A0 20 0.12

RPN1_HUMAN Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 59 0.11

ANXA2_HUMAN Annexin A2 55 0.1

G3P_HUMAN Glyceraldehyde-3-phosphate dehydrogenase 56 0.1

ACTB_HUMAN Actin, cytoplasmic 1 36 0.09 Protein Accession Protein Name Protein score emPAI

ILF2_HUMAN Interleukin enhancer-binding factor 2 36 0.09

ROA3_HUMAN Heterogeneous nuclear ribonucleoprotein A3 112 0.09

HNRDL_HUMAN Heterogeneous nuclear ribonucleoprotein D-like 66 0.08

HNRPF_HUMAN Heterogeneous nuclear ribonucleoprotein F 111 0.08

HNRPU_HUMAN Heterogeneous nuclear ribonucleoprotein U 103 0.08

PA2G4_HUMAN Proliferation-associated protein 2G4 26 0.08

EF1A1_HUMAN Elongation factor 1-alpha 1 55 0.07

GTR1_HUMAN Solute carrier family 2, facilitated glucose transporter member 1 25 0.07

HNRPK_HUMAN Heterogeneous nuclear ribonucleoprotein K 56 0.07

SEPT7_HUMAN Septin-7 37 0.07

TBA1B_HUMAN Tubulin alpha-1B chain 50 0.07

PTBP1_HUMAN Polypyrimidine tract-binding protein 1 127 0.06

DDX17_HUMAN Probable ATP-dependent RNA helicase DDX17 46 0.05

HNRPQ_HUMAN Heterogeneous nuclear ribonucleoprotein Q 102 0.05

HNRPR_HUMAN Heterogeneous nuclear ribonucleoprotein R 111 0.05 Protein Accession Protein Name Protein score emPAI

KU70_HUMAN ATP-dependent DNA helicase 2 subunit 1 43 0.05

NUCL_HUMAN Nucleolin 147 0.05

SFPQ_HUMAN Splicing factor, proline- and glutamine-rich 2 46 0.05

HXK1_HUMAN Hexokinase-1 44 0.04

IMMT_HUMAN Mitochondrial inner membrane protein 23 0.04

MA7D3_HUMAN MAP7 domain-containing protein 3 17 0.04

MATR3_HUMAN Matrin-3 102 0.04

MCM2_HUMAN DNA replication licensing factor MCM2 33 0.04

ERC6L_HUMAN DNA excision repair protein ERCC-6-like 32 0.03

POTEE_HUMAN POTE ankyrin domain family member E 36 0.03

SF3B1_HUMAN Splicing factor 3B subunit 1 46 0.03

U520_HUMAN U5 small nuclear ribonucleoprotein 200 kDa helicase 103 0.03

CHD4_HUMAN Chromodomain-helicase-DNA-binding protein 4 43 0.02

CLH1_HUMAN Clathrin heavy chain 1 73 0.02

CLH2_HUMAN Clathrin heavy chain 2 86 0.02 Protein Accession Protein Name Protein score emPAI

EPIPL_HUMAN Epiplakin 91 0.01

FLNA_HUMAN Filamin-A 30 0.01

PRKDC_HUMAN DNA-dependent protein kinase catalytic subunit 112 0.01 emPAI = Exponentially modified protein abundance index.

Supplementary Figure S1

Supplementary Fig S1. Expression of phospho (p)-ATM and p-DNA-PKcs in TSGH8301 cells under hypoxia. TSGH8301 cells were harvested after they had been exposed to hypoxia for 0, 1, 3, 6, 12, and 24 h, respectively. Equal amounts of protein (50 g) were loaded and assayed using Western blotting (A and B represent two dependent experiments). Supplementary Figure S2

Supplementary Fig S2. Nuclear translocation of RON in response to anticancer drugs. The formation of -H2AX foci and the nuclear translocation of RON in TSGH8301 cells were analyzed using confocal microscopy after the cells had been treated for 24 h with etoposide, doxorubicin, epirubicin, or mitomycin C.

Supplementary Figure S3

Supplementary Fig S3. Significance of RON on survival of TSGH8301 stable cells under hypoxia in the presence of etoposide or cisplatin. Supplementary Figure S4

Supplementary Fig S4. Colocalization of nuclear RON with the Ku70/DNA-PKcs complex in the presence of doxorubicin, epirubicin, or mitomycin C. The TSGH8301 stable cell lines overexpressing Vector control (TSGH8301/Vector) (A) or knockdown RON (TSGH8301/kdRON) (C) were used in this experiment. Each stable cell line was treated with anticancer drugs (etoposide, doxorubicin, epirubicin, or mitomycin C [as indicated]) for 24 h. After incubation, normal medium was replaced and treated with hypoxia for an additional 24 h. The images were captured using a confocal microscope.

SUPPLEMENTARY MATERIALS AND METHODS

Immunoprecipitation and trypsin digestion

The nuclear extract of hypoxic TSGH8301 cells was incubated with RON antibody (IgG as a control) overnight at 4C. The pull-down proteins were eluted with 1% SDS, alkylated with 50 mM of iodoacetamide (IAM) in the dark at room temperature for 30 min, and then concentrated using trichloroacetic acid. The precipitated proteins were resuspended with 50 mM of ammonium bicarbonate and digested by 50 g of trypsin for 18 h at 37°C.

LC-MS/MS identification

The ESI-MS data were obtained using a quadrupole time-of-flight (Q-TOF) micro-instrument (Micromass) equipped with a nanoflow high-performance liquid chromatography (HPLC) system (LC Packings). The digest solution (30 L) was injected onto a C18 nanoprecolumn cartridge (i.d. = 300 m  1 mm) (5 m C18, P/N160458; LC Packings) and then separated by a C18 column (i.d. = 75 m; o.d. = 280 m  15 cm) (3 m C18; LC Packings). The elution buffers were composed of mobile phase A: 0.1% formic acid in 2% acetonitrile (ACN) and mobile phase B: 0.1% formic acid in 80% ACN; a linear gradient from 5% to 90% for 60 min at a flow rate of 300 nL/min was used. The outlet of the column was connected to an electrospray ionization (ESI) tip using capillary tubing (i.d. = 20 m, o.d. = 280 m). For protein identification, the

MS/MS spectra were obtained using survey scans from m/z 400 to 1600, and the MS/MS scan was from m/z 50 to 2000; mass spectrometry software (MassLynx 4.0 Global ProteinLynx; Waters Corp., Milford, MA) was used to produce a peak list from raw data. All sequential scans with the same precursor ion were combined. The threshold to switch from MS to MS/MS was ten counts, and the switch from MS/MS back to MS was set up for signals that fell below three counts or after eight seconds. Proteins were identified using the MASCOT v2.2.1 search engine on the SwissProt_56 protein database (509019 sequences updated on Sep. 22, 2009). The mass tolerance was set at 0.2 Da for the precursor ions and 0.2 Da for the product ions. Carbamidomethyl (C) was chosen as a fixed modification, and one missed cleavage by trypsin was allowed. We set a cutoff score of 20 to eliminate low-score peptides and only “rank1” (Best match for each MS/MS) peptides were included. The candidate proteins were sorted by their exponentially modified protein abundance index (emPAI) calculated by dividing the number of sequenced peptides into total protein to estimate absolute protein abundance in proteomics (1) (Appendix Table S2).

References

1. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, et al. Exponentially modified protein abundance index (emPAI) for estimation

of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 2005;4:1265-72.