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Sema 3A Impacts Foxo 3a Signaling in Breast Cancer Progression

Explore the role of Sema 3A on Foxo 3a pathway dynamics in breast cancer cell lines. Investigate Sema 3A and its effect on tumor progression.

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Sema 3A Impacts Foxo 3a Signaling in Breast Cancer Progression

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

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

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

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

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

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

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

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

  9. Correlation between Sema 3A & pPTEN with breast cancer progression Supplementary Table S1 (Mishra et al )

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