Supplementary Figure Legends s8

SUPPLEMENTARY FIGURE LEGENDS

Figure S1. Sema 3A inhibits Akt phosphorylation and induces NRP-1-mediated and

PTEN-dependent FOXO 3a-DNA binding in MDA-MB-231 cells. (a) Serum starved cells were treated with Sema 3A for 45 min and level of p-Akt was analyzed by western blot. Akt served as loading control. (b) MDA-MB-231 cells were treated with Sema 3A

(100 ng/ml) for 0-240 min and FOXO 3a-DNA-binding was analyzed by EMSA. (c) Cells were treated with Sema 3A (100 ng/ml) alone or NRP-1 expression in these cells was inhibited by its siRNA, neutralizing antibody (5 µg/ml) or blocking peptide (20 µg/ml) and then treated with Sema 3A, nuclear extract was prepared and FOXO 3a-DNA-binding was analyzed by EMSA. (d) Cells were either treated with Sema 3A alone or individually transfected with PTENi, wt PTEN or dn PTEN and then treated with Sema 3A and FOXO

3a-DNA binding was performed as described above. (e) FOXO 3a-DNA-binding on

MelCAM promoter using wt and mut probes was analyzed by EMSA as described in

Materials and Methods. Excess unlabeled wt probe was used as positive control.

Figure S2. PTEN plays crucial role in Sema 3A-mediated MDA-MB-231 migration.

(a) MDA-MB-231 cells were either transiently transfected with wt PTEN, dn PTEN or

PTENi prior to Sema 3A treatment and used for migration assay. The migrated cells were stained and photographed. (b) Cells treated or transfected under similar conditions as described above were used for wound assay.Wound photographs were taken and analyzed.

Figure S3. FOXO 3a is involved in Sema 3A-mediated inhibition of breast cancer cell migration. (a, b) MDA-MB-231 cells were either treated with Sema 3A (100 ng/ml) or transfected with wt FOXO 3a, FOXO 3a TM or FOXO 3ai and then treated with Sema 3A and used for Boyden chamber and wound migration assays. The migrated cells in both assays were photographed. Figure S4. Blocking NRP-1 abrogates Sema 3A-mediated breast cancer cell migration. (a, b) MDA-MB-231 cells were either treated with Sema 3A (100 ng/ml) or

NRP-1 expression in these cells was inhibited by its siRNA, neutralizing antibody (5

µg/ml) or blocking peptide (20µg/ml) followed by Sema 3A treatment and used for Boyden chamber and wound migration assays. The migrated cells in both these assays were photographed.

Figure S5. Sema 3A regulates tumor-endothelial interaction through NRP-1 dependent paracrine mechanism. (a) Tumor-endothelial interaction was performed in

Boyden chamber or matrigel coated invasion chamber. NRP-1 expression in HUVECs was silenced either using its siRNA , neutralizing antibody (5 µg/ml) or blocking peptide (20

µg/ml) and were added to the upper chamber. CM collected from MDA-MB-231 cells treated with Sema 3A was used in the lower chamber. The invaded and migrated cells were stained and photographed. (b, c) NRP-1 expression in MDA-MB-231 cells was blocked using its siRNA or neutralizing antibody (5 µg/ml) followed by Sema 3A treatment and

CM was collected and used in lower chamber. HUVECs were added to upper chamber. The migrated and invaded HUVECs were stained, counted, analyzed statistically and represented graphically. Columns, means of three determinations; bars, s.e.m., **p and

##p<0.05 versus Sema 3A plus CM,*p and #p <0.05 versus Medium.

Figure S6. Sema 3A inhibits breast cancer cell migration. (a, b) Sema 3A overexpressing MDA-MB-231 cells were used for Boyden chamber and wound migration assays. (c) MCF-7 cells were treated with Sema 3A blocking peptide (20 µg/ml) for 24 h and subjected to wound assay. The photographs were captured.

Figure S7. Sema 3A suppresses VEGF-induced angiogenesis. (a, b) HUVECs (1x104 cells/well) were treated either with rh Sema 3A (100 ng/ml), VEGF (50 ng/ml) or in combination and used in tube formation assay. In separate experiments, cells were either

2 treated with NRP-1 neutralizing antibody (5µg/ml) or CM collected from MDA-MB-231 cells transfected with Sema 3A cDNA and then treated with VEGF. In other experiments, cells were pre-treated with NRP-1 neutralizing antibody followed by treatment with rh

VEGF and rh Sema 3A. The mean tube lengths were measured, analyzed statistically and represented as mean ± s.e.,*p<0.05 versus Medium and **p<0.05 versus VEGF. (c, d) In

CAM assay, gelatin sponge inserts were loaded with rh Sema 3A (100 ng/ml), rh VEGF

(100 ng/ml) or in combination and placed on the CAM of 4 day old fertilized white leg horn eggs. On day 6, photographs of CAMs were taken using stereo microscope equipped with camera and angiogenic response to various treatments was assessed and quantified as increase in vessel length and size and represented graphically. Error bars, s.e.m.,* p and

**p<0.05 versus VEGF.

Figure S8. Attenuation of endogenous Sema 3A augments breast tumor growth in

NOD-SCID mice and regulates expression of various tumor suppressive and angiogenic molecules. (a). MCF-7 or MCF-7-Sema 3A shRNA stable cells were injected into female NOD-SCID. After 7 weeks, tumors were dissected and photographed. (b)

MCF-7 cells were injected orthotropically into female NOD-SCID mice and Sema 3A blocking peptide (1 mg/Kg body weight) was injected into tumor sites. After 6 weeks, mice were sacrificed, dissected and photographed. (c) Immunohistochemical analyses of Sema

3A (green; i, ii), p-PTEN (red; iii, iv), p-FOXO 3a (red; v, vi), FOXO 3a (red; vii, viii) and

MelCAM (green; ix, x) in tumors obtained from mice injected with MCF-7 cells alone or

MCF-7 cells injected tumors treated with Sema 3A blocking peptide. (d)

Immunohistochemical analyses of Ki67 (red; i, ii), CD31 (red; iii, iv) and VEGF (red; v, vi) in same tumor sections as described in Supple Fig S8c. (e) Kaplan-Meier analysis of relapse free survival in breast cancer patients (n=2978) using KM plotter. Patients were

3 divided according to FOXO 3a expression (FOXO 3a; red line, positive; black line, negative).