Supplemental Figure S1

Transcription

1 o E p h re p h. 1 me p h. 2 me p h. 3 me p h. 4 h E p h. 4 me p h. 6 Supplemental Figure S1 2. No b b alone b + peptide bsorpt tion units N. anti-eph4 #V131 Ephrin2-P Tumor Meta astasis C seph4 (ng) D Eph receptors, fused to Fc (2ng) h-1 m-2 m-3 m-4 r-5 m-6 m-7 m-8 r-1 h-2 m-3 h-4 m-4 m-6 nti-eph4 #V265 nti-eph4 #V265

2 Supplemental Figure S2 Case #2 Case #3 Normal Tumor Normal Tumor Eph 2 Eph 2 Ep ph4 Ep ph4 Case #4 Case #5 Normal Tumor Normal Tumor Eph2 Eph2 Eph 4 Eph 4

3 Supplemental Figure S2 Eph4 levels Case ID Eph N T N T N T N T N T N T N T N T N T N T Normal Tumor Normal Tumor Normal Tumor Case #6 Case #7 Case #8 Case #9 Case #1 Case #11 Case #12 Case #13 Case #14 Case #15 Case #16 Case #17

4 Supplemental Figure S2 C Normal Liver Liver Metastasis Normal Liver Liver Metastasis Eph 2 Eph4 Case #19 Case #2 D Normal Colon Tumor Nodal Metastasis Eph h4 Case #21 Eph4 Eph2 N T M Eph 2

5 Supplemental Figure S2 E F Eph4 β-catenin Primary tumor Normal Tumor n Eph4 mrn Liver metastasis Fold difference i Matched tissue pairs

6 Supplemental Figure S3.5.1 Serum m-free.5 Fc Ephrin2/Fc (µg/ml) (µg/ml) : Eph4 IP: I: py I: Eph4 No primary α-eph4 1 + FITC rum-free Ser Fc Ephrin2/Fc (1µg/ml) (1µg/ml) (min) P: Eph4 I I: py I: Eph4 α-ephrin2 1 + PE-2 oth C Contro ol Ephrn 2-Fc Contro ol Ephrin n2-sirn GFP-s sirn Serum IP: Ep ph4 I: py I: Eph4 Ephrin2

7 Supplemental Figure S4 HT29 (Eph4+) Eph4-siRN (1nM) Time (h) Eph4 -H FL HT29 (Eph4+) Eph4 sirnδ Eph4 sirn g 1 1 Gillug FSC-H FSC-H FSC-H C 293T-hEph4 + Eph4 sirn D HT29 (Eph4+) E 12 HT29-parent HT29-mEph4 heph4 meph4 293T-parent 1nM 5nM 1nM 293T-mEph4 + Eph4 sirn 293T-pare ent 1nM 5nM 1nM Cell nu umber (% contro ol) Eph4 sirn (nm) mber (% contro ol) Cell nu SW62 (Eph4-) Eph4 sirn Eph4 sirnδ sirn (nm)

8 Supplemental Figure S4 F HT29 COLO-25 sirn (1nM) sirn (1nM) Eph4 Eph2 Ephrin2 Eph4 Ephrin2 Eph2 Eph4 Eph2 Ephrin2

9 Supplemental Figure S5 Tet off (ptt2) and G418 selection Tet regulated Eph4 sirn vector and puromycin selection Clonal selection and expansion Doxycycline present sirn not expressed; Eph4 expressed HT29 HT29 + TetR HT29 + TetR + sirn vector FF4 cells Doxycycline absent HT29-mock cells sirn expressed; Eph4 not expressed Eph4 shrn (11Kb) No RT con trol Doxycycline (ng/ml)

10 Supplemental Figure S6 Tetracycline + Tetracycline - FF4 Mock FF4 Mock HT29-parent Mock Tet Mock No tet FF4 Tet FF4 No tet

11 2 3 Supplemental Figure S7 hr 9 hr 12 hr l at hr) 12 hr 9 hr 12 hr 1 Eph4 sirnδ (5nM) Eph4 sirn (5nM) Cell-free area (% control Eph4 sirnδ Eph4 sirn (5nM) (5nM) Eph4 sirnδ (5nM) Eph4 sirn (5nM) s/hpf No. of cell Eph4 sirnδ (5nM) Eph4 sirn (5nM)

12 Supplemental Figure S8 bsorb bance (fold control) Eph4-siRN Eph4 sirnδ Caspase-8 Caspase bs sorbance (%co ontrol) Dose (nm) Eph4 sirn (nm) Eph4 sirnδ (nm) C Eph4 TRIL DR4 DR5 DcR1 Eph4-siRNΔ Eph4-siRN

13 Supplemental Figure S9 FF4 GFPsiRN TRILsiRN TRIL Lipo Cell nu umber (% contr rol) Tet GFP sirn TRIL sirn

14 Supplemental Figure S1 Eprhin2 C D Eph4-PE Eph4-Wt EC IC PM G C F F Eph4-PE Eph4-eGFP EC IC PM G C F F G F P Eph4-PE WT WT Eph4-PE WT Null Eph4 WT GFP Eph4-WT (~127KDa) Eph4-eGFP (~18 KDa) E Eph4 p-y eGFP Eph4 293-Eph4 293-Eph4- egfp IP: Eph4 293-eGFP 293-Eph4-eG GFP GFP GFP GFP GFP Eph4 293-eGFP 293-Eph4-eGFP bsorptio on units inding to Ephrin2-P 2.5 X 1^4 cells 5 X 1^4 cells 1 X 1^5 cells 3 X 1^5 cells 1.5 X 1^6 cells eGFP 293-Eph4 293-Eph4- egfp

15 Supplemental Figure S11 Cell number (% %control) SW48 cells (Eph4+) Eph4 sirnδ Eph4 sirn 1nM Eph4 sirn 2nM Cell number (% control) TRIL (ng/ml) Parent Empty vector Eph4-FL 1 Eph4-eGFP TRIL (ng/ml)