Supplementary Figure Legends Supplementary Fig. 1. Multiple five micron sections of liver tissues of rats treated with either vehicle (left; n=3) or CCl 4 (right; n=3) were co-immunostained for NRP-1 (green) and stellate cell markers, (A) desmin, (B) α-sma and (C) NG2. Representative images for each staining are shown in the left column for NRP-1 staining (in green), middle column for desmin or α-sma or NG2 (in red), and overlay of the two (in yellow) in the right column. Below each image is the colocalization analysis including Pearson s correlation for each of the overlay images. White bar over the overlay image represents the area selected for colocalization. Supplementary Fig. 2. (A) KDR expression was determined in LX2, hhsc, and endothelial cells (HUVEC) by immunoblotting (n=3). (B) KDR mrna presence was assessed by semiquantitative reverse transcript PCR from liver endothelial cells, HSC and LX2. β2 microglobulin (β2-mg) was used as PCR control. (C) hhsc treated with VEGF or PDGF were subjected to Western blotting with antibodies recognizing phospho-pdgfrβ and β-actin, used as loading control. (D) Immunoblot of hhscs treated alone or with combinations of the putative NRP-1 ligand, Sema3A and PDGF-BB ligand was probed with phospho-pdgfrβ and β-actin as loading control. (Representative autoradiographs or western blots are from three independent experiments). (E) Boyden chamber cell migration assay was conducted with hhsc using PDGF or Sema3A as the chemotactic
agent (n=3, p<.5). (F) Akt phosphorylation was assessed by Western blot by treating liver endothelial cells, HHSEC, or LX2 cells with VEGF at different time intervals. Total Akt and actin were used as internal loading control. While VEGF increased AKT phosphorylation in liver endothelial cells, AKT phosphorylation was not changed in response to VEGF in LX2. White line/box indicates splicing of non-contiguous samples. Supplementary Fig. 3. A. Left, human liver EC transfected with c-abl sirna were plated on growth factor reduced Matrigel, incubated with and without VEGF and tubulogenesis was measured after 24 hours. Right, human liver EC transfected with c-abl sirna or scramble sirna were lysed and c-abl knockdown was confirmed by Western blot. B. Cell migration was studied with human liver EC after knockdown of c-abl (p<.5). C. Human liver EC were assessed for c-abl activity in presence or absence of VEGF. c-abl activity was low in EC despite VEGF stimulation. VEGF positive control included p-akt which was increased in response to VEGF. Supplementary Fig. 4. A. Cell migration was studied with hhsc after knockdown of PDGFRβ using real time lapse videomicroscopy. Cellular distance traveled was measured by MetaMorph imaging software. PDGF-induced HSC migration was absent in PDGFRβ silenced cells despite presence of endogenous NRP-1 (n=3, p<.5). B. Cell migration was studied in LX2 with NRP-1 sirna mediated knockdown in the presence of FGF or PDGF. NRP-1 enhances FGF
induced migration in a statistically significant manner but to a quantitatively lower extent than that observed with PDGF. Western blot from lysates of LX2 cells transfected with sirna confirmed knockdown of NRP-1. Supplementary Fig. 5. A. The specificity of the binding of I 125 PDGF to cells was validated by using non-labeled PDGF (cold PDGF) to compete the labeled (hot PDGF). B. HSC incubated with NRP-1 sirna prior to incubation with PDGF ligand evidenced diminished binding to PDGF (n=3, p<.5). Supplementary Fig. 6. A. Protein lysates from human HSC, human liver EC, LX2 cells, and HepG2 cells were probed with NRP-1, PDGFR and actin antibody for Western blot analysis. B. Protein lysates from primary mouse HSC and primary mouse liver EC were probed with NRP-1 antibody for Western blot analysis. Arrows show the distinction of an additional higher molecular weight form of NRP-1 in human and mouse HSC. White line indicates splicing of a noncontiguous sample from the same membrane. Supplementary Fig. 7. HSC incubated with PDGF were harvested and lysed for co-immunoprecipitation assay using anti-phosphotyrosine antibody and then the precipitates were analyzed by SDS-PAGE and Western blot with PDGFR and p85 antibodies respectively.
Supplementary Fig. 8. Liver endothelial cells were plated on growth factor reduced Matrigel and incubated with VEGF and/or NRP-1b Ab and tubulogenesis was measured within each experimental group after 24 hours (n=3 separate experiments). Supplementary Fig. 9 Mouse HSC (mhsc) isolated from WT and NRP-1 f/f mice were used to study cell migration using Boyden Chamber with and without PDGF. mhsc from WT mice were pre-treated with NRP-1 antibody to study cell migration with and without PDGF. In parallel, mhsc from NRP-1 f/f mice were treated with Ad-Cre to delete NRP-1 from transduced cells. Ad-LacZ was used as a control. Depicted data are a juxtaposition of the data from Figure 4 and Figure 8 in order to allow side-by-side quantitative comparison of the effects of genetic and antibody based disruption of NRP-1 function on HSC migration.
Supplementary Fig. 1 Vehicle CCl 4 A NRP-1 Desmin Merge NRP-1 Desmin Merge 14 Intensity (AU) 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Pearson's Correlation Rr=.52 Intensity (AU) 12 8 6 4 2 1 3 5 7 9 11 13 15 17 19 Pearson's Correlation Rr=.47 B NRP-1 α-sma Merge NRP-1 α-sma Merge 2 Intensity (AU) 1 3 5 7 9 11 13 15 17 19 21 23 25 27 Pearson's Correlation Rr=.44 Intensity (AU) 1 3 5 7 9 11 13 15 17 19 21 23 25 27 Pearson's Correlation Rr=.45 C NRP-1 NG2 Merge NRP-1 NG2 Merge Intensity(AU) 16 12 8 4 1 4 7 1 13 16 19 22 25 28 Pearson's Correlation Rr=. Intensity (AU) 1 3 5 7 9 11 13 15 17 19 21 23 25 27 Pearson's Correlation Rr=.41
Control VEGF PDGF Supplementary Fig. 2 A B KDR β-actin IB: p-pdgfrβ β-actin HUVEC LX2 hhsc D IB: p-pdgfrβ C Control PDGF BB Sema 3A PDGF BB+Sema 3A β-actin 2 Number of cells migrated HHSEC LX2 VEGF(1ng/ml, min) 1 3 6 1 3 6 IB: p-akt IB: AKT IB: Actin Control PDGF-BB Sema-3A HHSEC hhsc LX2 HHSEC hhsc LX2 E KDR β2 MG F
Supplementary Fig. 3 A NS Tube Length (AU) 7 6 4 3 Control VEGF c-abl Actin Srcamble C-abl sirna Scrambled sirna c-abl sirna B % of cells migrated 18 16 14 12 8 6 NS Control VEGF C VEGF(1ng/ml, min) 3 IP: c-abl p-crk c-abl 3 lysates p-akt total-akt 4 2 Scrambled sirna c-abl sirna
Scrambled sirna PDGFR-β sirna PDGFR-β sirna Supplementary Fig. 4 A 2 PDGFRβ β-actin Scram ble sirna Scram ble sirna + PDGF PDGFRβ sirna PDGFRβ sirna+ PDGF Srcamble NRP-1 sirna Distance migrated (% of Control) B 2 NRP1 Control Actin FGF PDGF Scrambled sirna NRP-1 sirna % of cells migrated
Supplementary Fig. 5 A I125 PDGF I125 PDGF+cold PDGF (2 ng/ml) B Scramble sirna NRP-1 sirna Specific binding (ng/ml).9.8.7.6.5.4.3.2.1 -.1 1 161 62 1664.667 4-5 1 15 2 25 Concentration of I125 PDGF (ng/ml) 1/ Specific binding (ng/ml).7.6.5.4.3.2.1 -.1 5 1 15 2 25 Concentration of I125 PDGF (ng/ml)
EC (mouse) HSC(mouse) Supplementary Fig. 6 A. B. HepG2 (human) LX2 (Human) HSC (Human) EC (Human) IB NRP-1 PDGFR Ab: NP-1(Goat anti-rat) Actin NRP-1
Supplementary Fig. 7 IP: p-tyrosine PDGF - + - + PDGFR P85 RFP NRP1 LX2
Supplementary Fig. 8 Tube length (% of control) 16 14 12 8 6 4 2 Control NRP1-Ab Control- VEGF NRP1- Ab-VEGF
Supplementary Fig. 9 2 % of cells migrated Vehicle PDGF Vehicle NRP-1 antibody Ad-GFP Ad-Cre WT mhsc NRP-1 f/f mhsc