Different responses of colorectal cancer cells to

Different responses of colorectal cancer cells to alternative sequences of cetuximab and oxaliplatin Elli Narvi , Katri Vaparanta , Anna Karrila , Deepankar Chakroborty , Sakari Knuutila , 1

1,2

1,2

1,2

3

Arto Pulliainen , Maria Sundvall , and Klaus Elenius* 1

1,4

1,4

1

Institute of Biomedicine and Medicity Research Laboratories, University of Turku, Finland

2

Turku Doctoral Programme of Molecular Medicine, Turku, Finland

3

Department of Pathology, Haartman Institute, University of Helsinki, Finland

4

Department of Oncology, Turku University Hospital, Turku, Finland

Supplementary materials and methods

Apoptosis prediction model. To create the apoptosis prediction model, the HCA7-derived RNAseq data from genes from the Reactome pathways R-HSA-109606 and R-HSA-5357769 and the experimental annexin V apoptosis data were used as a training set for a polynomial support vector machine. The trained model was used to predict the amount of apoptosis in the RKO and DLD-1 cell lines from the RNAseq data. The sensitivity of each gene in the model was determined by setting the mean gene expression value for each treatment sequentially for each gene one at a time and running it through the prediction algorithm.

A

EGFR mAb (µg/ml)

% of control cells (MTT)

0.01

0.1

1

10

EGFR mAb (µg/ml) 100

0.01

0.1

1

10

HCA7

100

100

DLD-1

80

oxaliplatin

60

cetuximab

40

panitumumab

20 0 0.1

1

10

100

1000

0.1

oxaliplatin (µM)

1

10

100

1000

oxaliplatin (µM)


B DLD-1

HCA7

100 80

oxaliplatin

60

oxaliplatin + cetuximab (10 µg/ml)

40 20 0 0.1

1

10

100

oxaliplatin (µM)

1000

0.1

1

10

100

1000

oxaliplatin (µM)

Supplementary Figure 1. Administration of EGFR mAbs and oxaliplatin alone and in simultaneous combination. The sensitivity of CRC cell lines to a 72 hour treatment with the indicated concentrations of EGFR mAbs, oxaliplatin, or their combination was tested by MTT cell viability assays. A) The EGFR mAb-sensitive HCA7 (left panel) and the EGFR mAb-insensitive DLD-1 (right panel) cells were treated with the EGFR mAbs cetuximab or panitumumab, or oxaliplatin as single agents. B) The EGFR mAb-sensitive HCA7 (left panel) and the EGFR mAb-insensitive DLD-1 (right panel) cells were treated with a simultaneous combination of cetuximab and oxaliplatin. Data are expressed as the relative MTT signal compared to untreated control cells. Mean +/– SD is shown.


HCA7

100

DLD-1

80 60 40 20 0 0

0.1

1

10

irinotecan (µM)

100

1000

0

0.1

1

10

100

1000

irinotecan (µM) 1st cetuximab (10 µg/ml) 2nd irinotecan irinotecan 1st irinotecan 2nd cetuximab (10 µg/ml)

Supplementary Figure 2. Sequential administration of cetuximab and irinotecan. Sequential administration of cetuximab before or after irinotecan was compared to administration of irinotecan alone with MTT assays of CRC lines. HCA7 (A) and DLD-1 (B) cells were 1) treated for 24 hours (for day 3 of the experiment) with the indicated concentrations of irinotecan alone (black curves), 2) treated first for 48 hours (days 1 and 2) with 10 μg/ml cetuximab followed by 24 hour (day 3) treatment with the indicated concentrations of irinotecan (red curves), or 3) treated first for 24 hours (day 3) with irinotecan followed by 48 hour (days 4 and 5) treatment with 10 μg/ml cetuximab (green curves). MTT analysis was carried out after day 5 of the experiment. The mean +/– SD are shown.

DLD-1 HCA7 RKO DLD-1 HCA7 RKO DLD-1 HCA7 RKO DLD-1 HCA7 RKO DLD-1 HCA7 RKO DLD-1 HCA7 RKO

REACTOME

1st drug: – 1st drug (h): 24 2nd drug: – 2nd drug (h): 0

cet 24 – 0

ox 24 – 0

– 24 ox 6

cet 24 ox 6

ox 24 cet 6 PLC-gamma1 signalling R-HSA-167021 SHC-related events triggered by IGF1 R R-HSA-2428933 p130Cas linkage to MAPK signaling for integrins R-HSA-372708 SHC1 events in ERBB4 signaling R-HSA-1250347 SHC1 events in EGFR signaling R-HSA-180336 Frs2-mediated activation R-HSA-170968 Signalling to p38 via RIT and RIN R-HSA-187706 ARMS-mediated activation R-HSA-170984 Hedgehog ligand biogenesis R-HSA-5358346 Signaling by NOTCH3 R-HSA-1980148 Regulation of Apoptosis R-HSA-169911 CDK-mediated phosphorylation and removal of Cdc6 R-HSA-690 17 Signaling by Leptin R-HSA-2586552 Cyclin E associated events during G1 ;S transition R-HSA-69202 Rho GTPase cycle R-HSA-194840 Gamma carboxylation R-HSA-163841 G alpha (i) signalling events R-HSA-418594 Signaling by Retinoic Acid R-HSA-5362517 E2F mediated regulation of DNA replication R-HSA-11 3510 Signaling by PDGF R-HSA-186797 Phospholipase C-mediated cascade; FGFR1 R-HSA-5654219 Asparagine N-linked glycosylation R-HSA-446203 Negative regulation of FGFR1 signaling R-HSA-5654726 Signaling by ERBB2 R-HSA-1227986 FRS-mediated FGFR1 signaling R-HSA-5654693 Spry regulation of FGF signaling R-HSA-1295596 Regulation of IGF transport and uptake R-HSA-381426 Signaling by Activin R-HSA-1502540 Cellular response to heat stress R-HSA-3371556 Integrin alphaIIb beta3 signaling R-HSA-354192 PLC-gamma1 signalling R-HSA-167021 Caspase activation via extrinsic apoptotic signaling pathway R-HSA-5357769 DAG and IP3 signaling R-HSA-1489509 Translocation of GLUT4 to the plasma membrane R-HSA-14451 48 SHC1 events in ERBB2 signaling R-HSA-1250196 PLCG1 events in ERBB2 signaling R-HSA-1251932 EGFR downregulation R-HSA-182971 GO and Early G1 R-HSA-1538133 Activated NOTCH1 transmits signal to the nucleus R-HSA-2122948 Cellular response to hypoxia R-HSA-22627 49 RHO GTPases Activate Formins R-HSA-5663220 Mitotic Prophase R-HSA-68875 Eukaryotic Translation Termination R-HSA-72764 Nucleosome assembly R-HSA-774815 Formation of the beta-catenin;TCF transactivating complex R-HSA-201722 DNA Damage;Telomere Stress Induced Senescence R-HSA-2559586 Chromosome Maintenance R-HSA-73886 RHO GTPases activate PKNs R-HSA-5625740 Oxidative Stress Induced Senescence R-HSA-2559580 Peptide ligand-binding receptors R-HSA-375276 PI3K;AKT activation R-HSA-198203 Regulation of mitotic cell cycle R-HSA-453276 G2/M DNA damage checkpoint R-HSA-69473 TGF-beta receptor signaling in EMT R-HSA-2173791 Degradation of beta-catenin by the destruction complex R-HSA-195253 IRS activation R-HSA-74713 Energy dependent regulation of mTOR by LKB1-AMPK R-HSA-380972 NGF processing R-HSA-167060 Nuclear signaling by ERBB4 R-HSA-1251985 Mitotic Metaphase and Anaphase R-HSA-2555396 Unfolded Protein Response (UPR) R-HSA-381119 p53-independent DNA Damage Response R-HSA-6961 0 Ubiquitin-dependent degradation of Cyclin D1 R-HSA-69229 Degradation of DVL R-HSA-4641258 Chemokine receptors bind chemokines R-HSA-3801 08 GRB2 events in EGFR signaling R-HSA-179812 GRB2/SOS provides linkage to MAPK signaling for Integrins R-HSA-354194 Chaperonin-mediated protein folding R-HSA-390466 Hedgehog off state R-HSA-561 0787 Class B;2 (Secretin family receptors) R-HSA-373080 Signaling by SCF-KIT R-HSA-1433557 Base-Excision Repair; AP Site Formation R-HSA-73929 Lagging Strand Synthesis R-HSA-69186 NRAGE signals death through JNK R-HSA-193648 PKB-mediated events R-HSA-1 09703 Centrosome maturation R-HSA-380287 Intrinsic Pathway for Apoptosis R-HSA-1 09606 Class C;3 (Metabotropic glutamate;pheromone receptors) R-HSA-420499

Supplementary Figure 3. RNAseq gene expression analysis by pathways annotated using Reactome database. DLD-1, HCA7, and RKO cells were subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: 1) control medium for 24 hours (white bar), 2) cetuximab for 24 hours (blue bar), 3) oxaliplatin for 24 hours (left black bar), 4) control medium for 24 hours followed by oxaliplatin for 6 hours (right black bar), 5) cetuximab for 24 hours followed by oxaliplatin for 6 hours (red bar), or 6) oxaliplatin for 24 hours followed by cetuximab for 6 hours (green bar). Gene expression was analyzed using RNAseq. Data were sectioned into pathway matrices according to the annotations of the Reactome database and converted into a heatmap.


WIKIPATHWAYS


cet 24 – 0

ox 24 – 0

– 24 ox 6

cet 24 ox 6

ox 24 cet 6 Telomere Maintenance WP1928 Nucleosome assembly WP1 874 Mitotic Prophase WP2654 Translocation of GLUT4 to the plasma membrane WP2m Regulation of Hypoxia-inducible Factor (HIF) by ·oxygen WP2727 IL-4 Signaling Pathway WP395 Wnt Signaling Pathway WP428 TCR Signaling WP1927 Mitotic G1-G1 :S phases WP1858 Kinesins WP1842 M;G1 Transition WP2785 EPO Receptor Signaling WP581 Degradation of beta-catenin by the destruction complex WP2773 IL-2 Signaling Pathway WP49 Apoptosis WP254 Regulation of toll-fike receptor signaling pathway WP1449 DNA Damage Response WP707 Rac1:Pak1:p38:MMP-2 pathway WP3303 Interleukin-1 Induced Activation of NF-kappa-B WP3656 Nephrin interactions WP1867 Nonsense-mediated Decay (NMD) WP2710 Growth hormone receptor signaling WP2657 Triacylglyceride Synthesis WP325 Processing of Capped Intronless Pre-mRNA WP1890 Parkin-Ubiquitin Proteasomal System pathway WP2359 Signaling by NGF WP1976 Signaling by NOTCH2 WP2718 Gap junction traffickina and regulation WP1820 Energy metabolism WP1541 POUSF1 (OCT4): SOX2: NANOG activate genes related to proliferation WP3333 Interferon type I Signaling pathways WP585 Signaling by FGFR1 WP3335 IL-9 Signaling Pathway WP22 TNFR2 non-canonical NF-kB pathway WP3398 Mtitotic Metaphase and Anaphase WP2757 NIK-->noncanonical NF-kB Signaling WP3545 Sterol Regulatory Element-Binding Proteins (SREBP) Signaling WP1982 Mtetabolism of water-soluble vitamins and cofactors WP1857 Cell junction organization WP1793 Transcription factor regulation in adipogenesis WP3599 Cytosolic sensors of pathogen-associated DNA WP2794 tRNA Aminoacylation WP1938 Signaling by Rho GTPases WP1917 Interferon alpha:beta Signaling WP1835 DNA Damage Response (only ATM dependent) WP710 GPCR downstream Signaling WP1824 Potassium Channels WP2669 Nonhomologous End-Joining (NHEJ) WP3550 BDNF Pathway WP2152 Generic Transcription Pathway WP1822 SIRT1 negatively regulates rRNA Expression WP3364 Nuclear Receptors in Lipid Metabolism and Toxicity WP299 GPCRs: Other WP117 Extracellular vesicle-mediated Signaling in recipient cells WP2870 Transport of inorganic cations: anions and amino acids:oligopeptides WP1936 L1CAM interactions WP1843 EBV LMP1 Signaling WP262 TNFs bind their physiological receptors WP3350 DNA Damage:Telomere Stress Induced Senescence WP3565 Histone Modifications WP2369 Oxidation by Cytochrome P450 WP43 Aryl Hydrocarbon Receptor Pathway WP2873 Sudden Infant Death Syndrome (SIOS) Susceptibility Pathways WP706 Degradation of the extracellular matrix WP2774 Signaling by FGFR4 WP3334 Signaling by FGFR3 WP3336 Selenium Metabolism and Selenoproteins WP28 Assembly of the primary cilium WP3498 Interactome of polycomb repressive complex 2 (PRC2) WP2916 Circadian rythm related genes WP3594 Monoamine GPCRs WP58 Cell Differentiation- meta WP2.023 Cell Differentiation- Index WP2029 DNA Double Strand Break Response WP3543 Cell Cycle Checkpoints WP1775 DNA methylation WP3359 NAD Biosynthesis II (from tryptophan) WP2485 Vitamin D Receptor Pathway WP2877 SREBF and miR33 in cholesterol and lipid homeostasis WP2011 Vitamin B12 Metabolism WP1533 Folate Metabolism WP176 C-type lectin receptors (CLRs) WP3354 Gamma carboxylation: hypusine formation and arylsulfatase activation WP2762 GPVI-mediated activation cascade WP1826 Signal amplification WP1908 Signaling by ERBB4 WP2781 Cholesterol biosynthesis WP1795 Histidine: lysine: phenylalanine: tyrosine: proline and tryptophan catabolism WP3573 Fatty Acid Biosynthesis WP357 Ubiquinol biosynthesis WP2734 MAP kinase activation in TLR cascade WP2792 TP53 Regulates Metabofic Genes WP3349 Signaling of Hepatocyte Growth Factor Receptor WP313 Copper homeostasis WP3286 Costimulation by the CD28 family WP1799 Assembly of collagen fibrils and other multimeric structures WP2798 RANKL:RANK (Receptor activator of NFKB (liagnd)) Signaling Pathway WP2018 NGF Signaling via TRKA from the plasma membrane WP1873 Regulation of Microtubule Cytoskeleton WP2.038 Fanconi Anemia Pathway WP3569 Signaling by Activin WP2791 Mitochondrial translation WP331 0 RNA Polymerase II Transcription WP1906 Fas Ligand (FasL) pathway and Stress induction of Heat Shock Proteins (HSP) regulation WP314 Binding and Uptake of Ligands by Scavenger Receptors WP2784 Signaling by PDGF WP1916 Senescence and Autophagy in Cancer WP615 MAPK targets: Nuclear events mediated by MAP kinases WP1845 Oncostatin M Signaling Pathway WP2374 Quercetin and Nf-kB: AP-1 Induced Cell Apoptosis WP2435 Mitochondrial biogenesis WP3331 RIG-I:MDA5 mediated induction ·of IFN-alpha:beta pathways WP1904 APC:C-mediated degradation of cell cycle proteins WP3557 Branched-chain amino acid catabolism WP3556 G13 Signaling Pathway WP524 Focal Adhesion WP306 Integrin-mediated Cell Adhesion WP185 Integration of energy metabolism WP1831 TGF-beta Receptor Signaling WP560 Signaling by NOTCH1 WP2720 Notch Signaling Pathway WP61 G1 to S cell cycle control WP45 Cell Cycle WP179 Trans-Golgi Network Vesicle Budding WP3578 Nucleotide-binding domain: leucine rich repeat containing receptor (NLR) signaling pathways WP2763 Cellular response to heat stress WP3395 NRF2 pathway WP2884 Wnt Signaling Pathway and Pluripotency WP399 MAPK6:MAPK4 Signaling WP3307 PDGF Pathway WP2526 Brain-Derived Neurotrophic Factor (BDNF) Signaling pathway WP2380 IL-3 Signaling Pathway WP286 RHO GTPases Activate WASPs and WAVEs WP3388 Kit receptor Signaling pathway WP304 ErbB Signaling Pathway WP673 MAP3K8 (TPL2)-dependent MAPK1:3 activation WP3576 Fatty acid: triacylglycerol and ketone body metabolism WP1817 P1 Metabolism WP2747 Nuclear Receptors Meta-Pathway WP2882 Mitotic Prometaphase WP2652 RHO GTPases Activate Formins WP3379 Nucleotide Excision Repair WP1980 Oxidative Stress WP408 GPCR ligand binding WP1825 Class I MHC mediated antigen processing & presentation WP3577 Arachidonic acid metabolism WP2650 Peptide GPCRs WP24 G Protein WP3577 Pathways WP35 Oxidative phosphorylation WP623 miRNA targets in ECM and membrane receptors WP2911

Supplementary Figure 4. RNAseq gene expression analysis by pathways annotated using Wikipathways database. DLD-1, HCA7, and RKO cells were subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: 1) control medium for 24 hours (white bar), 2) cetuximab for 24 hours (blue bar), 3) oxaliplatin for 24 hours (left black bar), 4) control medium for 24 hours followed by oxaliplatin for 6 hours (right black bar), 5) cetuximab for 24 hours followed by oxaliplatin for 6 hours (red bar), or 6) oxaliplatin for 24 hours followed by cetuximab for 6 hours (green bar). Gene expression was analyzed using RNAseq. Data were sectioned into pathway matrices according to the annotations of the Wikipathways database and converted into a heatmap.


BIOCARTA


cet 24 – 0

ox 24 – 0

– 24 ox 6

cet 24 ox 6

ox 24 cet 6 DEATH_PATHWAY BIOPEPTIDES_PATHWAY GH_PATHWAY MTA3_PATHWAY IL2RB_PATHWAY MAPK_PATHWAY AT1R_PATHWAY EDG1_PATHWAY IL22BP PATHWAY INTEGRIN_PATHWAY EGF _PATHWAY PDGF _PATHWAY IL3_PATHWAY MET_PATHWAY CREB_PATHWAY CSK_PATHWAY BARRESTIN_SRC_PATHWAY AGPCR_PATHWAY TRKA_PATHWAY CBL_PATHWAY PTDINS_PATHWAY ATRBRCA_PATHWAY SODD_PATHWAY EIF _PATHWAY CDC42RAC_PATHWAY PROTEASOME_PATHWAY SRCRPTP PATHWAY NFKB_PATHWAY RAC1_PATHWAY TID_PATHWAY TNFR2_PATHWAY EGFR_SMRTE_PATHWAY BARRESTIN_PATHWAY MAL_PATHWAY GLYCOL YSIS_PATHWAY P27 _PATHWAY PTC1_PATHWAY IL 1R_PATHWAY IL7 _PATHWAY CDK5_PATHWAY PLCE_PATHWAY CHEMICAL_PATHWAY GATA3_PATHWAY RARRXR_PATHWAY CALCINEURIN_PATHWAY EPHM _PATHWAY IL 10_PATHWAY CELL2CELL_PATHWAY TFF _PATHWAY EPO_PATHWAY ERK_PATHWAY MCALPAIN_PATHWAY WNT_PATHWAY INSULIN_PATHWAY NGF _PATHWAY SPRY_PATHWAY

Supplementary Figure 5. RNAseq gene expression analysis by pathways annotated using Biocarta database. DLD-1, HCA7, and RKO cells were subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: 1) control medium for 24 hours (white bar), 2) cetuximab for 24 hours (blue bar), 3) oxaliplatin for 24 hours (left black bar), 4) control medium for 24 hours followed by oxaliplatin for 6 hours (right black bar), 5) cetuximab for 24 hours followed by oxaliplatin for 6 hours (red bar), or 6) oxaliplatin for 24 hours followed by cetuximab for 6 hours (green bar). Gene expression was analyzed using RNAseq. Data were sectioned into pathway matrices according to the annotations of the Biocarta database and converted into a heatmap.

A

B

Intrinsic Pathway for Apoptosis (Reactome)

Caspase Activation via Extrinsic Apoptotic Signaling Pathway


cet 24 – 0

2

PMAIP1 YWHAG

1

– 24 ox 6

cet 24 ox 6

ox 24 cet 6


BAK1

3

Variability between cell lines

ox 24 – 0



(Reactome)

BAD

0

BID

-1

BMF

BCL2 E2F1 MAPK8 DYNLL2 PPP3CC YWHAH CASP7 BCL2L1 NMT1 TFDP1 CYCS YWHAQ BBC3 CASP8 GZMB AKT1 XIAP DYNLL1CASP3 DIABLOYWHAZ APAF1 PPP3R1

CASP9

-2

0

YWHAB YWHAE

– 24 ox 6

cet 24 ox 6

ox 24 cet 6

FASLG

1

TICAM1 LY96

APPL1

CASP8

CFLAR TLR4 TLR3 UNC5A TNFSF10 MAGED1 TRADD RIPK1 TICAM2 CASP9 TNFRSF10A CASP3

DAPK1

-1

FADD

TNFRSF10B UNC5B

-2

-3 -2

-1

0

1

2

-2

-1

Sensitivity score

0

1

2

Sensitivity score

C

D

measured apoptosis Cell line: 5

predicted apoptosis

HCA7

DLD-1

measured apoptosis

RKO

DLD-1

***

RKO

** 10 8

4

Apoptosis (%)

ox 24 – 0

2

BCL2L11 BAX

cet 24 – 0

3 1

6 4 2

0

0

2

control cetuximab

** 20

oxaliplatin

10

1st cetuximab, 2nd oxaliplatin

0

1st oxaliplatin, 2nd cetuximab

-1 -2

E BCL2

600

YWHAQ

6

BID

80

550

4

90

extrinsic pathway FASLG

0

60 0

50

BBC3

450 20

40 15 20

APAF1

120

40

APPL1

CASP8

CYCS 45

30

100 40 80 60

ox 1 al 2n st c ipla d et tin ox ux 1s alip ima 2n t o la b, d xa tin ce li tu pl xi ati m n, ab ox 1 al 2n st c ipla d et tin ox ux 1s alip ima 2n t o la b, d xa tin ce li tu pl xi ati m n, ab ox 1 al 2n st c ipla d et tin ox ux 1s alip ima 2n t o la b, d xa tin ce li tu pl xi ati m n, ab

10

FADD

80

60

500

120 100

0.5

70

2

0

1

35

25 20

ox 1 al 2n st c ipla d et tin ox ux 1s alip ima 2n t o la b, d xa tin ce li tu pl xi ati m n, ab ox 1 al 2n st c ipla d et tin ox ux 1s alip ima 2n t o la b, d xa tin ce li tu pl xi ati m n, ab

Relative mRNA expression

F

intrinsic pathway

Supplementary Figure 6. Gene expression changes in RNAseq analysis predict apoptotic response for "cetuximab after oxaliplatin". Apoptosis measured by annexin V analysis of the HCA7 cells (Fig. 2B) and the expression of apoptosis-related genes in two Reactome-annotated pathways (A, B) were used to create a model to predict apoptosis for the two other cell lines, DLD-1 and RKO, included in the RNAseq analysis (C). D, Apoptosis measured by annexin V analysis of DLD-1 and RKO cells. **, P < 0.01. E and F, box plot presentations of the 10 genes that best predicted the measured apoptosis of HCA7 cells and were subsequently used to model the apoptosis of DLD-1 and RKO cells as shown in panel C. The box plots indicate the median (thick horizontal line), the second and third quartiles (the box), and the range (whiskers) of the data.

15 5

10

pERK1/2

pAKT

pEGFR

pp38a

pJNK1/2/3

15

15

10

10

5

5

0

0

10

5

5 0

0

pSTAT2

pSTAT6

pSTAT3

0

pCREB

5 15 5

10

5

Phosphorylation (abritrary densitommetric units)

5 0

0

pSRC

0

0

pYES

pFYN 5

5

pLCK

pLYN

5

5 5

0

0

pp53

0

pCHK2

15

20

10

15

0

pGSK3a/b

0

pFAK

pβ-catenin

5

5

0

0

5

10 5

5 0

0

pPRAS40 25

pAMPKa1 10

15

pHSP27

pAMPKa2 10

5

5

10 5

5 0

0

0

0

1s

tc 1s etux t o im xa a lip b, o con la 2n xal tro tin d ip l , 2 ox lat nd ali in ce pla tu tin xi m ab 1s tc 1s etux t o im xa a lip b, o con la 2n xal tro tin d ip l , 2 ox lat nd ali in ce pla tu tin xi m ab 1s tc 1s etux t o im xa a lip b, o con la 2n xal tro tin d ip l , 2 ox lat nd ali in ce pla tu tin xi m ab 1s tc e 1s tux t o im xa a lip b, o con la 2n xal tro tin d ip l , 2 ox lat nd ali in ce pla tu tin xi m ab 1s tc e 1s tux t o im xa a lip b, o con la 2n xal tro tin d ip l , 2 ox lat nd ali in ce pla tu tin xi m ab

0

20 15

5

10

pHSP60 25

10

20

0

Supplementary Figure 7. Phosphoarray analysis of sequential treatments. HCA7 cells were subjected to different regimens containing 10 μg/ml cetuximab and/or 50 μM oxaliplatin: 1) control medium for 24 hours (white), 2) control medium for 24 hours followed by oxaliplatin for 1 hour (black), 3) cetuximab for 24 hours followed by oxaliplatin for 1 hour (red), or 4) oxaliplatin for 24 hours followed by cetuximab for 1 hour (green). Phosphorylation profiles were analyzed using Proteome Profiler Human Phospho-Kinase Array Kit and quantified by densitometry. Mean +/– range is shown.

Cell line:

HCA7

Apoptosis (%)

* 10

no drugs thymidine

8

oxaliplatin

6 4

1st thymidine, 2nd oxaliplatin

2 0

Supplementary Figure 8. Effect of G1 arrest by thymidine on oxaliplatin-induced apoptosis. HCA7 cells were arrested in G1 by 8 hour treatment with or without 2 mM thymidine followed by 18 hour treatment with or without 50 μM oxaliplatin. Apoptosis was measured by annexin V analysis. Mean +/– SD is shown for three independent experiments. *, P < 0.05.

Blots in Fig. 4B 700 nm

800 nm

actin

pERK

actin

ERK

actin

pSTAT3

actin

STAT3

Merged kDa 245 180 135 100 75 63 48 35 25 20 17 11 kDa 245 180 135 100 75 63 48 35 25 20 17 11 kDa 180 135 100 75 63 48 35 25 20 17 11

kDa 245 180 135 100 75 63 48 35 25 kDa20 245 180 135 100 75 63 48 35 25 20 17 11

actin

cleaved PARP

actin

p53

kDa 245 180 135 100 75 63 48 35 25 20 17 11

p21

kDa 245 180 135 100 75 63 48 35 25 20 17 11

Blots in Fig. 4D 700 nm STAT3 actin

kDa 245

700 nm pSTAT3

800 nm

Merged

180 135 100 75 63 48 35 25 20 17 11

Supplementary Figure 9. Full length blots of figure 4B and 4D.

kDa 245 180 135 100 75 63 48 35 25 20 17 11