Supplementary Figure 1

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1 Supplementary Figure SEM4C PLXN2 8 SEM4C C 3 Cancer Cell Non Cancer Cell Expression log2 ratio Expression p value.1 D Supplementary Figure 1. Expression of Sema4C and Plexin2 in breast cancer and normal tissues. () ioinformatic analysis of data retrieved from GENT ( indicating that both SEM4C and PLXN2 mrn levels are upregulated in human breast cancers (n=2635), compared to normal breast (n=91). Each circle represents an individual sample. The Y-axis indicates expression values normalized to target density of 5 as mentioned in GENT. () Graphical representation of the Log2 ratio between Sema4C levels in matched tumor vs. adjacent normal tissue pairs (n=43), from the analysis of expression dataset GSE15852; p<.1. (C) Graphical representation of normalized Sema4C expression levels in immortalized breast cancer cell lines and non-tumoral cell lines, based on bioinformatic analysis of public dataset ( (D) Kaplan-Meier analysis of overall patient survival (OS; follow-up at 12 years) of unselected breast cancer patients (n=142) correlation to Plexin2 (PLXN2) high and low expression subgroups, based on online dataset analysis (by p=. 75.

2 Supplementary Figure 2 mrn levels (fold change) SEM4C mrn levels (fold change) PLEXIN2 114kDa 45kDa Sema4C ctin 24kDa 45kDa Plexin2 ctin C 14kDa Plexin 2 45kDa ctin D Cell growth (bs 595nm) shcontrol Cell growth (bs 595nm) shcontrol shplexin2-1 shplexin Days Days Supplementary Figure 2. Sema4C/Plexin2 signaling blockade in breast cancer cells causes growth arrest. () qrt-pcr analysis of the mrn expression levels of Sema4C and Plexin2 in cells upon silencing by two different shrns. () Immunoblotting analysis of Sema4C and Plexin2 in gene-silenced cells (same as previous panel); actin provided a protein loading control. (C) Immunoblotting analysis revealing the expression of Plexin2 dominant negative construct (2-DN) in transduced cells, with Plexin2- specific antibodies; actin provided a protein loading control. (D) Cell growth curves of cells transduced with two different shrn constructs targeting Sema4C and Plexin2 (same cells described in panels and ). Data are the mean ± SD from three separate experiments; p<.1.

3 Supplementary Figure 3 MD-M 231 Time: Hours 12 Hours 24 Hours 48 Hours Plexin 2-DN shplexin 2 shcontrol Supplementary Figure 3. Sema4C/Plexin2 signaling blockade impairs tumor cell growth Time-lapse microscopy analysis of MD-M-231 cells subjected to Sema4C or Plexin2 silencing, or expressing the Plexin2 dominant negative construct (2-DN). Representative images are snapshots taken every 12-hour intervals. Scale bar: 1 µm. The full movie is separately provided in Supplemental Material.

4 Supplementary Figure 4 shctrl shctrl MD-M 231 Crystal Violet C shctrl CFSE Dye-Green DID Dye-Red Dapi (Nuclei)-lue D CFSE Dye-Green DID Dye-Red Dapi (Nuclei)-lue E Percentage of cells analyzed (%) multinucleated cells Supplementary Figure 4. Sema4C/Plexin2 signaling blockade causes cytokinesis defects. () right field image of MD-M-231 and cells either subjected to Sema4C silencing or controls. () Microscopy images of MD-M-231 described above stained with crystal violet. Scale bars: 1 μm. (C)Immunofluorescence images of mixed population of MD-M-231 cells labelled with either DID dye(far red) or CFSE(Green) and treated them with shrn(control) and analysed by immunofluorescence for fused multinucleated cells(yellow). Nuclei were stained with DPI.(D) Immunofluorescence images of mixed population of MD-M-231 cells labelled with either DID dye(far red) or CFSE(Green) and subjected to Sema4C-silencing and analysed by immunofluorescence for fused multinucleated cells(yellow). Nuclei were stained with DPI. Images from three representative fields are shown.(e) Quantification of percentage of Sema4C silenced cells which are multinucleated and CFSE positive(green) or DID positive(far red), or fused (Yellow); n>5 cells in three independent experiments.scale bar: 1 µm.

5 Supplementary Figure 5 MD-M 231 shctrl shplxn2 Plexin2-DN Invasion Migration Supplementary Figure 5. Sema4C/Plexin2 signaling blockade impairs migration and invasion. Representative images of Transwell assays assessing the migration and matrix invasion of MD-M- 231 cells subjected to Sema4C or Plexin2 silencing, or expressing Plexin2-DN, compared to shctrl cells. Magnification: 1x.

6 Supplementary Figure 6 P21 DPI P53 DPI H1152 DMSO Supplementary Figure 6. Rho-kinase inhibition promotes nuclear accumulation of p21 and p53 tumor suppressor proteins in cells. Representative images of immunofluorescence analysis of p21 and p53 tumor suppressor proteins distribution (in green) in cells treated with Rho-dependent kinase inhibitor H1152 (2μm) or DMSO-vehicle for 5 days. Nuclei are stained with DPI (blue). Scale bar: 1 µm.

7 Supplementary Figure 7 mrn levels (Fold Change) Plxn2 24kDa 45kDa Plexin2 ctin C IP-Erb2 IP-Erb2 185kDa I-P-Erb2 185kDa I-P-Erb2 185kDa I-Erb2 185kDa I-Erb2 24kDa I-Plexin 2 24kDa I-Plexin 2 Supplementary Figure 7. Enhanced Sema4C signaling promotes Erb2 phosphorylation. ()Graphic representation of the fold change in mrn levels of Plexin 2 in cells stably expressing Sema4C-secr, compared to control cells, as determined by qrt PCR analysis and Western blot analysis of Plexin2 expression in cells stably overexpressing Sema4C-secr or Mock; ctin served as protein loading control.() cells were treated with the indicated concentrations of recombinant Sema4C or mock, for 2 minutes and protein lysates were immunopurified with anti-erb2 antibody, followed by immunoblotting to reveal tyrosine phosphorylation and interaction with Plexin 2; total Erb2 staining provided an internal reference.(c) Protein lysates of cells stably expressing Sema4C-secr or Mock were immunopurified with anti-erb2 antibody, followed by immunoblotting to reveal tyrosine phosphorylation and interaction with Plexin 2; total Erb2 staining provided an internal reference.

8 Supplementary Figure 8 MOCK Sema4C-secr MOCK Sema4C-secr Y DMSO E-Cadherin eta-catenin Mock +DMSO Sema4C-secr +DMSO Sema4C-secr +Y Relative Migration (fold change) Sema4C Y Supplementary Figure 8. Phenotypic reversion of Sema4C-overexpressing cells upon inhibition of Rho- or Erb2-signaling. () Representative immunofluorescence images showing the distribution of E-cadherin or eta-catenin (in green) in MCF7 cells overexpressing Sema4C (or mock), treated for 24 hours with Rho-kinase inhibitor (Y , 1μM), or DMSO. Nuclei were stained with DPI. Scale bar: 1 µm. () Transwell assays assessing the migration of cells stably expressing Sema4C (or mock), treated with Rho-kinase inhibitor Y (1μM) or DMSO. Quantification of migration (fold changes) is shown on the right; data are the mean ± SD from three independent experiments; p<.1. Magnification: 1x.

9 Supplementary Figure 9 ER positive patients Supplementary Figure 9. High Sema4C expression is associated with poor relapse-free survival in estrogen receptor positive breast cancer patients. ()Kaplan-Meier analysis of relapse-free survival (OS; follow-up at 12 years) of estrogen receptor positive (ER+) patients (n=1983) correlation to Sema4C high and low expression subgroups, based on online dataset analysis (by p=.74.