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Supplementary Figure S1 (Mishra et al) MDA-MB-231 Time (min) 0 30 45 90 180 240 Sema 3A - + + + + + (100 ng/ml) Foxo 3a Free probe 1 2 3 4 5 6 b Foxo 3a.

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Presentation on theme: "Supplementary Figure S1 (Mishra et al) MDA-MB-231 Time (min) 0 30 45 90 180 240 Sema 3A - + + + + + (100 ng/ml) Foxo 3a Free probe 1 2 3 4 5 6 b Foxo 3a."— Presentation transcript:

1 Supplementary Figure S1 (Mishra et al) MDA-MB-231 Time (min) 0 30 45 90 180 240 Sema 3A - + + + + + (100 ng/ml) Foxo 3a Free probe 1 2 3 4 5 6 b Foxo 3a Free probe MDA-MB-231 - - + + + + - + - - - - - - - - + - - - - - - + - - - + - - Sema 3A (100 ng/ml) Coni PTENi dnPTEN wt PTEN 1 2 3 4 5 6 d - - + + + + - + - - - - - - - + - - - - - - + - - - - - - + Sema 3A (100 ng/ml) Coni NRP1i NRP1 neu Ab (5µg/ml) NRP1 block pep (20 µg/ml) 1 2 3 4 5 6 Foxo 3a Free probe MDA-MB-231 c Time (min) 0 5 10 15 30 45 Sema 3A - + + + + + (100 ng/ml) MDA-MB-231 1 2 3 4 5 6 Akt Mw (kDa) pAkt 64 50 a 64 50 Cold Probe-1 (25X) Foxo 3a Free probe MDA-MB-231 FP Wt Probe-1 FP Mut Probe-2 e 1 2 3 4 5

2 Control Coni Sema 3A Sema A + PTENi Sema 3A + dn PTEN Sema 3A + wt PTEN MDA-MB-231 0 h 16 h b MDA-MB-231 a a Control Coni b c Sema 3A Sema 3A + wt PTEN d Sema 3A + PTENi e f Sema 3A + dn PTEN Supplementary Figure S2 (Mishra et al)

3 b Control Coni Sema 3A Sema 3A+ wt Foxo 3a Sema 3A + Foxo 3ai Sema 3A + Foxo 3a TM MDA-MB-231 0 h 16 h Supplementary Figure S3 (Mishra et al) a ab c d e f Control Coni Sema 3A Sema 3A + wt Foxo 3a Sema 3A + Foxo 3ai Sema 3A + Foxo 3a TM MDA-MB-231

4 Supplementary Figure S4 (Mishra et al) a MDA-MB-231 Control a b Coni Sema 3A c Sema 3A + NRP1i d Sema 3A + NRP1 neu Ab e Sema 3A + NRP1 block pep f ControlConi 0 h Sema 3A Sema 3A + NRP1i Sema 3A+ NRP1 neu Ab Sema 3A + NRP1 block pep 16 h MDA-MB-231 b

5 Co-migration HUVEC (Upper chamber) Coni Medium rhSema 3ArhSema 3A + NRP-1i rhSema 3A + NRP-1 neu Ab CM MDA-MB-231 cells (Lower chamber) Co-invasion a b c d e a b cd e I II b Supplementary Figure S5 (Mishra et al) ConiMedium NRP1i NRP1 neu Ab NRP1 block pep Co-migration HUVEC (Upper chamber) a c e f Co-invasion CM MDA-MB-231 cells (Lower chamber) a b c d e f b d a I II HUVEC Medium Coni CM MDA-MB-231 + rh Sema 3A (Lower chamber) NRP-1 neu Ab NRP-1i migration/invasion (% Control) ** * * # ## CM MDA-MB-231 + rh Sema 3A c

6 b MDA-MB-231 MDA-MB-231- Sema 3A 0 h 16 h Supplementary Figure S6 (Mishra et al) 0 h 24 h MCF-7 Control Sema 3A block pep (20μg/ml) c a b MDA-MB-231 MDA-MB-231- Sema 3A a

7 a rhVEGF + NRP1 neu Ab rhVEGF + rhSema 3A + NRP1 neu Ab rhVEGF + CM MDA-MB-231 -Sema 3A Medium a rhVEGF b rhSema 3A c rhVEGF + rhSema 3A d e g f b Tube length (% Control) * ** VEGF (50 ng/ml) Medium NRP1 neu Ab (5 µg/ml) Sema 3A (100 ng/ml) CM MDA-MB-231-Sema 3A - + - + + + + + - - - - - - - - - - + + - - - + + - + - - - - - - - + Medium rhVEGF rhSema 3A rhVEGF + rhSema 3A 0 h 48 h a b c d ef g h c Change in length and size * ** Medium rhVEGF rhSema 3A rhVEGF + rhSema 3A d Supplementary Figure S7 (Mishra et al)

8 Supplementary Figure S8 (Mishra et al) d Ki67 i ii CD31/DAPI iii iv VEGF MCF-7- Sema 3A block pep MCF-7 vi v pFoxo 3a MCF-7-Sema 3A shRNA MCF-7-mock vector a MCF-7-Sema 3A block pep (1 mg/Kg body weight) MCF-7 b MCF-7- Sema 3A block pep MCF-7 MelCAM ixx Sema 3A c i ii pPTEN iii iv Foxo 3a vii viii v vi Probability FOXO3A Relapse free survival (years) n=2878 e

9 Test Statistics a Sema 3A ExpressionpPTEN Expression Mann-Whitney U.000 Wilcoxon W 1.000 Z Asymp. Sig. (2-tailed).317 Exact Sig. [2*(1-tailed Sig.)] 1.000 b a. Grouping Variable: SrNo b. Not corrected for ties. Correlation between Sema 3A & pPTEN with breast cancer progression Supplementary Table S1 (Mishra et al )

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