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. Author manuscript; available in PMC: 2013 Jun 25.
Published in final edited form as: Pigment Cell Melanoma Res. 2010 Nov 17;24(1):165–174. doi: 10.1111/j.1755-148X.2010.00797.x

Plexin B1 inhibits integrin-dependent pp125FAK and Rho activity in melanoma

Lindy McClelland 1, YuLin Chen 1, Joanne Soong 2, IHsin Kuo 2, Glynis Scott 1,2
PMCID: PMC3692372  NIHMSID: NIHMS464714  PMID: 21029396

Summary

Semaphorins are secreted and membrane bound proteins that regulate axon guidance through receptors Plexins and neuropilins. Plexin B1, the Semaphorin 4D receptor, is a recently described tumor suppressor protein for melanoma. We recently showed that Plexin B1 abrogates activation of the oncogenic receptor, c-Met, by its ligand, hepatocyte growth factor (HGF), in melanoma. We have now investigated the effect of Plexin B1 on integrin-dependent pp125FAK activation, and the small GTP-binding protein Rho, in melanoma. Integrin receptors and Rho play critical roles in melanoma progression, through regulation of migration, proliferation and apoptosis. We engineered two human melanoma cell lines expressing Plexin B1 and analyzed integrin-dependent migration, integrin-dependent pp125FAK activation, and Rho activity. Results show that Plexin B1 abrogates integrin-dependent migration and activation of pp125FAK. We also show that Rho activity is significantly reduced in cells expressing Plexin B1, and that Plexin B1 suppresses HGF-dependent Rho activation.

Keywords: melanoma, semaphorin, plexin, integrin, Rho

Introduction

Plexin receptors are a family of highly conserved transmembrane proteins, which alone or in cooperation with neuropilins, transduce effects of semaphorins (Castellani and Rougon, 2002). Semaphorins are secreted or membrane bound proteins originally described in the nervous system, but are also expressed in lung, kidney, bone and lymph tissue (Fujisawa, 2004; Moretti et al., 2006; Potiron et al., 2009; Takegahara et al., 2006). The best characterized effects of Plexin receptor signaling are on remodeling of the actin cytoskeleton (Bagnard, 2001; Korostylev et al., 2008; Nakamura et al., 2000). Plexin receptors from each of the four Plexin families regulate cell adhesion, cell migration, axon guidance, and neuronal pathfinding (Nakamura et al., 2000; Negishi et al., 2005). The downstream targets of Plexins include integrins, Rho GTP-binding proteins, cofilin and Ras, which control multiple facets of cell behavior, including cytoskeletal remodeling and cell migration.

Loss or gain of Plexins and their cognate ligands, as well as mutations in Plexin receptors, are described in a variety of malignancies (Neufeld et al., 2005). Plexin B1, the receptor for semaphorin 4D, is mutated in prostate carcinoma (Wong et al., 2007), and its expression is down-regulated in renal cell carcinoma (Gomez Roman et al., 2008) and breast carcinoma (Rody et al., 2007). In breast carcinoma, a recent study has shown that loss of Plexin B1 is strongly correlated with poor prognosis (Rody et al., 2009). Our laboratory is interested in the role of Plexin receptors in melanocyte and melanoma biology. Melanocytes are derived from the neural crest, and migrate to the skin, where they reside in the basal epidermal layer. Although the genesis of melanoma is complex, melanomas are thought to arise from a stepwise progression of loss or gain of tumor suppressor and tumor promoter proteins, along with epigenetic changes, resulting in invasion, migration and metastasis. Metastatic spread of melanoma is the principle cause of morbidity and mortality associated with this disease. Our previous work showed that Plexin B1 expression is lost in melanoma in vivo, particularly in deeply invasive and metastatic tumors (Stevens et al., 2010). In combination with work showing that introduction of Plexin B1 into human melanoma cell lines abrogates metastasis in a mouse model (Argast et al., 2009), the data support a role for Plexin B1 as a tumor suppressor protein for melanoma. Although the mechanism by which loss of Plexin B1 promotes melanoma progression remains to be fully defined, we identified the c-Met receptor as a downstream target of Plexin B1 in melanoma (Stevens et al., 2010). Introduction of Plexin B1 into human melanoma cell lines significantly inhibited activation of the c-Met receptor by its ligand, hepatocyte growth factor (HGF). Because c-Met is frequently activated in metastatic melanomas, suppression of c-Met signaling is a potential mechanism by which Plexin B1 suppresses late stages of melanoma progression (Halaban et al., 1993).

Integrins are heterodimeric proteins composed of α and β chains that bind extracellular matrix proteins, such as fibronectin, collagen, laminin and vitronectin (Arnaout et al., 2007). Integrins link the cell membrane to the extracellular matrix through attachment of the cytoplasmic tail of the β chain to cytoskeletal associated proteins such as talin and α–actinin. Integrin activation results in pleiotropic effects including cell migration, invasion through basement membranes, and resistance to apoptosis (Reddig and Juliano, 2005). Integrin activation stimulates the phosphorylation and activation of focal adhesion kinase (pp125FAK; Mitra et al., 2005) which controls several integrin-dependent processes, including cell migration, and cell survival (Tomar and Schlaepfer, 2009). The Rho family of small GTP-binding proteins includes Rho, Rac and Cdc42. The principle effect of Rho proteins is on the actin cytoskeleton, with each protein having a well defined role in the formation of stress fibers (Rho), lamellipodia (Rac) and filopodia (Cdc42) in virtually all cells studied (Hall et al., 1993; Heasman and Ridley, 2008). Rho proteins alternate between active (GTP-bound) and inactive (GDP-bound) forms. In addition to effects on cytoskeletal remodeling, signaling by Rho proteins impacts cell proliferation, gene expression, and apoptosis, through signaling to diverse downstream targets (Ridley, 2001). Integrins and Rho share multiple downstream targets and are cross-activating or inhibiting, depending upon the cell type (Barry et al., 1997; Hotchin and Hall, 1995).

Our prior investigation into signaling pathways of Plexin B1 in melanoma suggest that inhibition of c-Met signaling is an important mechanism by which Plexin B1 suppresses melanoma progression (Stevens et al., 2010). Constitutive signaling of c-Met stimulates pp125FAK and Rho activation, leading to tumor progression (To and Tsao, 1998). Further, increased expression and activation of pp125FAK and Rho are described in many malignancies, including melanoma, independent of c-Met receptor activation (Golubovskaya et al., 2009; Hao et al., 2009; Lydolph et al., 2009; Narumiya et al., 2009; Routhier et al., 2010; Timar et al., 1997). In this report we have studied two human metastatic melanoma cell lines (YUSIK and YUMAC), engineered to express human Plexin B1, to examine effects of Plexin B1 on integrin-dependent pp125FAK activation and Rho activity. YUSIK and YUMAC melanoma cells do not show constitutive activation of the c-Met receptor, which permits identification of downstream targets of Plexin B1, independent of its suppressive effects on c-Met. Our data suggest that Plexin B1 has multiple targets that contribute to its role as a tumor suppressor protein for melanoma.

Results

Plexin B1 suppress migration of YUSIK melanoma towards fibronectin, but not invasion through a basement membrane

Migration and invasion are key processes in melanoma metastasis. To assess the effect of Plexin B1 on migration of melanoma towards fibronectin, YUSIK cells were placed in basal medium for 18 h and then seeded in the upper well of a 96 well migration chamber. The bottom wells contained basal medium with fibronectin, or basal media alone. Cells were allowed to migrate for 24 h (Figure 1A). Cells expressing empty vector (“LacZ”) and cells expressing Plexin B1 (“PBA”) showed dose-dependent migration towards fibronectin. The rate of migration was significantly higher (P < 0.001) at a fibronectin concentration of 50 μg/ml. The addition of recombinant Sema4D did not affect migration of PBA cells (data not shown), consistent with prior reports that the receptor signals constitutively (Conrotto et al., 2004; Giordano et al., 2002; Stevens et al., 2010). To determine if Plexin B1 abrogates invasion through a basement membrane, cells were placed in the top well of a matrigel invasion chamber and allowed to invade for 48 h (Figure 1B). There was no significant difference in invasion of Plexin B1 expressing cells, compared with LacZ controls (P = 0.16). Similar results were obtained when invasion was carried out for 24 h (data not shown).

Figure 1.

Figure 1

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Plexin B1 inhibits migration of melanoma towards fibronectin. (A) Shown is quantitative analysis (relative fluorescence units, rfu) of PBA and LacZ migration towards fibronectin over a 24-h time period. LacZ and PBA cells migrated towards fibronectin in a dose-dependent manner, however, migration of LacZ cells was higher at all concentrations of fibronectin, compared with PBA cells. The numbers above the bars indicate the fold change in migration at each fibronectin concentration, compared with migration towards basal media without added fibronectin (“nt”; no treatment). The difference in migration between LacZ and PBA cells was statistically significant at a fibronectin concentration of 50 μg/ml (P < 0.001). Each column represents the averaged results of nine separate wells, ±SD. Results are representative of two experiments. (B) Shown is quantitative analysis (relative fluorescence units, rfu) of PBA and LacZ cells that invaded through a basement membrane substrate over a 48-h time period. No significant difference in invasion was detected between LacZ and PBA cells (P = 0.16). Each bar represents the average fluorescence intensity from 3 separate experiments, from duplicate wells ±SD.

Plexin B1 abrogates integrin-dependent activation of pp125FAK

pp125FAK is a non-receptor tyrosine kinase localized to focal contacts, and was originally identified as a tyrosine phosphorylated protein in pp60v-src transformed cells (Kanner et al., 1990). Auto-phosphorylation of pp125FAK occurs upon integrin activation, and is closely correlated with its' kinase activity (Hanks et al., 2003). We examined levels of active (phosphorylated) pp125FAK in response to adhesion to fibronectin, an integrin-dependent process, in LacZ and PBA cells. Serum-starved cells were plated on poly-L-lysine, which does not activate integrins, or fibronectin, for 1 h. Unattached cells were rinsed off, and the remaining cells were lysed and blotted for active pp125FAK. Figure 2A shows a representative Western blot from three individual experiments; Figure 2B shows densitometry analysis of active pp125FAK normalized to total pp125FAK. Activation of pp125FAK in response to attachment to fibronectin was significantly higher (P < 0.05) in LacZ compared with PBA cells. In LacZ cells, active pp125FAK increased an average of 2-fold when attached to fibronectin, compared with PBA cells, in which active pp125FAK was increased by 1.4-fold. Similar experiments were performed in the second human meta-static melanoma cell line (YUMAC) engineered to express the Plexin B1 receptor. Because this cell line attached poorly to poly-L-lysine, activation of pp125FAK was only tested in cells attached to fibronectin. Figure 2C shows Western blots from two experiments; Figure 2D show densitometry analysis in which active pp125FAK is normalized to total pp125FAK. Following attachment to fibronectin, YUMAC LacZ cells showed a 1.4 fold increase in active pp125FAK compared with 1B2 cells, consistent with suppression of pp125FAK activity by Plexin B1. Levels of β1-integrins were similar in LacZ and PBA cells, as analyzed by FACS analysis (data not shown). Because levels of pp125FAK may be increased in melanoma, we determined levels of mRNA of pp125FAK in YUMAC and YUSIK melanoma cells expressing Plexin B1, or empty vector, using comparative real time PCR (Figure 2E). The fold difference in pp125FAK message between YUSIK LacZ and Plexin B1 expressing clones and YUMAC LacZ and Plexin B1 expressing clones was calculated using the Pfaffl equation (Pfaffl, 2001) with β-actin as a normalizing gene, and was 0.45 (±/‒0.1 SD) and 0.22 (±/‒ 0.03 SD), respectively. Because a fold change of at least 1.3 is generally accepted to represent a significant change in gene expression (Dirisi et al., 1996; Huggins et al., 2008; Schena et al., 1996), we conclude that levels of pp125FAK are not regulated by Plexin B1 in these cell lines. These data suggest that Plexin B1 suppresses pp125FAK activation in melanoma, but does not regulate expression.

Figure 2.

Figure 2

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Plexin B1 inhibits integrin-dependent pp125FAK activation in melanoma. (A) One hour after attachment to fibronectin (100 μg/ml), robust activation of pp125FAK was detected in LacZ cells. In contrast, PBA cells showed less activation of pp125FAK following attachment to fibronectin, compared with LacZ cells. Both cell types showed constitutive activity of pp125FAK, as shown by active pp125FAK in cells plated on poly-L-lysine. Shown is a representative Western blot from three separate experiments in which total cell lysates were resolved on 10% SDS-PAGE. (B) Densitometry analysis of Western blots from three separate experiments are shown. Phosphorylated pp125FAK was normalized to total pp125FAK. Columns represent the averaged density of bands (arbitrary units) from three experiments, ±SD. Active pp125FAK was significantly higher (P < 0.05) in LacZ cells compared with PBA cells when cells were attached to fibronectin. (C) Results from two experiments are shown in which YUMAC LacZ and 1B2 Plexin B1 expressing clones were allowed to attach to fibronectin (100 μg/ml) for 1 h. Robust activation of pp125FAK was detected in LacZ cells. In contrast, 1B2 cells showed less activation of pp125FAK following attachment to fibronectin, compared with LacZ cells. Shown are 10% SDS-PAGE in which total cell lysates were resolved. (D) Densitometry analysis of each experiment is shown. Phosphorylated pp125FAK was normalized to total pp125FAK. Columns represent the average density of each band (arbitrary units). (E) PCR products from one of 4 separate experiments for pp125FAK and β-actin were resolved on 1% agarose gel. A band corresponding to the expected molecular weight of pp125FAK was observed in YUSIK and YUMAC melanoma cells. No difference in levels of pp125FAK PCR product were observed between LacZ and Plexin B1-expressing clones, which was confirmed by comparative real time PCR.

Plexin B1 suppresses Rho activity in melanoma

Rho is a critical signaling protein involved in regulation of the actin cytoskeleton, and expression and activation of Rho are increased in a variety of malignancies, including melanoma (Sanz-Moreno and Marshall, 2009). When Rho is active, cells displays actin stress fibers, which span the length of the cell and aid in cellular adhesion. Cells with prominent stress fibers are generally highly spread out and elongated. We analyzed stress fibers in YUSIK and YUMAC cells expressing empty vector and Plexin B1 by staining serum starved cells with fluor-594-conjugated phalloidin (Figure 3A). Immunofluorescence microscopy of stained cells showed that stress fibers are barely detectable in Plexin B1 expressing cells, compared with LacZ controls. The presence of stress fibers in LacZ cells, in the absence of growth factors, suggests that Rho is constitutively active in these cell lines.

Figure 3.

Figure 3

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Plexin B1 inhibits the activity of Rho in melanoma. (A) Shown are representative images of PBA, LacZ (YUSIK) and 1B2, LacZ (YUMAC) cells under phase contrast microscopy (bar = 10 μ), grown in complete media, and immunofluorescence microscopy of cells that were plated on multiwall dishes, and placed in basal media overnight, followed by stained for actin using phalloidin conjugated with Alexa-fluoro-594 (bar = 50 μ). Actin stress fibers (arrow) are visualized in LacZ cells, whereas cells expressing Plexin B1 contain few stress fibers. (B) GTP-bound Rho was measured in LacZ and cells expressing Plexin B1 in YUSIK and YUMAC cells. Shown is the amount of GTP-bound Rho as ng/μg/total cellular protein (×102). Melanoma cells expressing Plexin B1 showed significantly lower levels (approximately 50%, P < 0.05) active Rho, compared with LacZ cells. Each column represents the averaged results of 4 experiments (LacZ and PBA) or three experiments (LacZ and 1B2) in which samples were measured in duplicate, ±SD. (C) GTP-bound Rho was measured in YUSIK LacZ and PBA cells in basal medium following treatment with hepatocyte growth factor (HGF) (10 ng/ml). Shown is the amount of GTP-bound Rho as ng/μg/total cellular protein (×102). Levels of GTP-Rho are higher in LacZ compared with PBA cells (2.64 ± 0.12 vs. 1.59 ± 0.10 ± SD respectively) in the absence of HGF. Treatment with HGF stimulated an increase in GTP-Rho at 10 and 30 min in LacZ cells. In contrast, HGF had no effect on levels of GTP-Rho at either time points in PBA cells. Each column represents the average of triplicate wells ±SD. The results are representative of two separate experiments.

We measured levels of active Rho in serum starved cells using a Rho G-LISA assay, which measures GTP bound Rho. Similar results were obtained in YUMAC and YUSIK cell lines (Figure 3B). YUSIK LacZ cells expressed 2.33 ng/ug total protein (×102) GTP-bound Rho compared with PBA cells, which expressed 1.0 ng/ug total protein (×102) GTP-bound Rho. The difference in GTP-Rho was significant (P = 0.013, n = 4 separate experiments). YUMAC LacZ cells expressed 2.54 ng/ug total protein (×102) GTP-bound Rho, compared with 1B2 cells, which expressed 1.3 ng/ug total protein (×102) GTP-bound Rho. The difference in GTP-Rho was significant between YUMAC LacZ and 1B2 cells (P = 0.025, n = 3 separate experiments).

Hepatocyte growth factor, also known as scatter factor, binds to and activates the c-Met receptor, resulting in activation of Rho (Takaishi et al., 1994). We have shown that Plexin B1 suppresses activation of the c-Met receptor, and migration in response to HGF (Stevens et al., 2010). To determine if Plexin B1 suppresses HGF-dependent activation of Rho, YUSIK cells were serum starved overnight and then treated with HGF (10 ng/ml) and GTP-bound Rho was measured (Figure 3C). Basal levels of GTP-Rho were 40% higher in LacZ compared with PBA cells. HGF stimulated a 16 and 40% increase in GTP-Rho in YUSIK LacZ cells at 10 and 30 min of treatment, respectively. In contrast, HGF had no effect on levels of GTP-bound Rho in cells expressing Plexin B1.

Discussion

The Plexin receptors belong to the c-Met family of scatter factor receptors, based on a conserved 500 amino acid “Sema domain” that is present in the extracellular region of the Plexin and c-Met receptors (Negishi et al., 2005). Plexin B1 is a non-tyrosine kinase receptor with a well-described function as a GTPase protein for R-Ras (Oinuma et al., 2004; Perrot et al., 2002; Saito et al., 2009). Previous reports show that Plexin B1 is a tumor suppressor protein for melanoma, and that Plexin B1 suppresses activation of the oncogenic c-Met receptor (Argast et al., 2009; Stevens et al., 2010). In this report we examined the effect of Plexin B1 on integrin-dependent pp125FAK activation, and Rho activity, in two human melanoma cell lines engineered to express the receptor. We chose human metastatic melanoma cell lines in which c-Met is not constitutively active, in order to identify targets of Plexin B1 that are independent of its suppressive effects on c-Met receptor signaling. Our data show that Plexin B1 inhibits integrin-dependent pp125FAK activity, and suppresses basal levels of active Rho in both cell lines. These results point to new downstream targets of Plexin B1 in melanoma that may contribute to its function as a tumor suppressor protein.

Activation of pp125FAK by integrin engagement is an important step in migration, because pp125FAK regulates the assembly of focal contact proteins at the leading edge of the migratory cell (Parsons et al., 2000). Constitutive p125FAK activation, or increased expression, has been linked to melanoma progression, however, it is generally accepted that aberrant pp125FAK activation, rather than increased expression, is more commonly seen in melanoma (Akasaka et al., 1995; Kahana et al., 2002; Scott and Liang, 1995; Vink et al., 1993). While we found slight variability in pp125FAK expression between LacZ and Plexin B1 expressing cells at the protein level, we did not detect consistent differences in levels of pp125FAK, and no evidence that pp125FAK is regulated at the message level. We did observe significant inhibition of integrin-dependent pp125FAK activation, however, as demonstrated by analysis of phosphorylated pp125FAK in cells attached to fibronectin in melanoma cell lines engineered to express Plexin B1. The significance of this finding will require further study, as the effects of expression of Plexin B1 on migration towards fibronectin were quite modest. Because migration is a complex process, it is likely that the effects of Plexin B1 on integrin activation are not sufficient to robustly suppress migration. However, pp125FAK activation suppresses apoptosis in cells detached from a cell matrix (“anoikis”; Gilmore, 2005), and promotes vasculogenic mimicry in melanoma (Hess et al., 2005). It is possible that the suppression of pp125FAK activity by Plexin B1 suppresses aggressive behavior in melanoma through regulation of apoptosis or new vessel formation, in addition to its' effects on migration. Further studies will be needed to test this notion.

Melanoma and melanocyte migration towards fibronectin is integrin dependent (Scott et al., 1992; Tian et al., 2007). Plexin B1 inhibits integrin activity in other cell types (Oinuma et al., 2006). Interestingly, R-Ras activates integrins in melanoma (Gawecka et al., 2010). Because Plexin B1 is a GTPase activating protein for R-Ras, loss of Plexin B1 in melanoma may increase integrin affinity or avidity through enhanced R-Ras activation. While our data suggest that Plexin B1 suppresses integrin activation in melanoma, additional experiments will be needed to define the effect of Plexin B1 on integrin activation in melanoma.

Rho is a multifunctional protein that regulates assembly of the actin cytoskeleton, but also controls cell adhesion, motility, migration, and gene expression (Ridley, 1997). Plexin B1 receptor signaling regulates the activity of Rho, resulting in either activation or inhibition, depending upon the cell type (Barberis et al., 2005; Korostylev et al., 2008, Perrot et al., 2002). Because activation of Rho promotes melanoma progression (Ruth et al., 2006) our data suggest that Plexin B1 dependent suppression of Rho may contribute to the tumor suppressor effects of Plexin B1. Analysis of stress fibers, and measurement of GTP-Rho, showed that Plexin B1 suppresses basal levels of Rho activity in two melanoma cell lines by approximately 50%. Rho is a downstream target of c-Met activation (Takaishi et al., 1994). When cells were treated with HGF, LacZ cells showed the expected increase in Rho activation, whereas cells expressing Plexin B1 showed no detectable Rho activation. Whether the lack of Rho activation in response to HGF is due to suppressive effects of Plexin B1 on c-Met receptor activity, or to suppression of Rho through pathways independent of c-Met, remains to be determined. Experiments in which c-Met receptor is silenced, or activation is blocked with specific antagonists of receptor activation, will be required to answer this question. The functional significance of Rho suppression by Plexin B1 in melanoma remains to be defined. We attempted to determine if Rho contributes to migration or invasion of melanoma, however, inclusion of an inhibitor of the Rho downstream effector ROCK, did not detectably affect invasion of cells across a basement membrane, or migration of LacZ or PBA cells towards fibronectin (unpublished data). The well documented role of Rho in the progression of cancer, as well as melanoma, suggests that suppression of Rho by Plexin B1 could be a potentially important mechanism by which Plexin B1 functions as a tumor suppressor protein for melanoma, however further studies will be needed to confirm this.

In summary, we have identified two additional targets of Plexin B1 signaling in melanoma, independent of its effects on the c-Met receptor; integrin-dependent pp125FAK activation, and Rho activity, in two melanoma cell lines engineered to express the receptor. We found a modest, yet significant effect, of Plexin B1 on migration towards fibronectin, however, because of the myriad effects of pp125FAK activation on variety of cell processes, it is reasonable to propose that Plexin B1-dependent pp125FAK suppression may affect other cellular processes. In combination with the known effects of Plexin B1 on suppression of c-Met receptor activity, a complex picture emerges of the signaling pathway of Plexin B1 in melanoma. In cells driven by c-Met receptor activation, Plexin B1 is expected to suppress melanoma progression through inhibition of c-Met dependent downstream targets, including Rho and pp125FAK. Because Plexin B1 suppresses pp125FAK and Rho activity, independent of its effects on the c-Met receptor, Plexin B1 is expected to be a powerful suppressor of pp125FAK and Rho in melanoma cells driven by constitutive c-Met activation. However, a subset of melanomas do not show aberrant activity of c-Met, and in those melanomas, the ability of Plexin B1 to inhibit integrin-dependent pp125FAK activation, and Rho activity, through pathways distinct from suppression of c-Met, suggests that Plexin B1 potentially functions as a tumor suppressor protein in a heterogeneous population of melanomas.

Methods

Reagents

Rabbit polyclonal antibodies to β-actin and mouse monoclonal antibodies against RhoA were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies, mouse IgG2 antibodies, fibronectin from bovine plasma, hepatocyte growth factor (HGF) and soybean trypsin inhibitor were purchased from Sigma Co., (St Louis, MO, USA). Antibodies against focal adhesion kinase (pp125FAK) phosphorylated on Tyr 397 were purchased from Millipore (Temecular, CA, USA). Antibodies against total pp125FAK were purchased from MBL (Nagoya, Japan). Fullrange rainbow molecular weight marker was purchased from Amersham Life Sciences (Arlington Heights, IL, USA). Fluoro-594-conjugated phalloidin was obtained from Molecular Probes Inc (Portland, OR, USA). Rho-GLISA assay was purchased from Cytoskeletal Inc. (Denver, CO, USA). Poly-L-Lysine was purchased from R&D systems (Minneapolis, MN, USA).

Cells and cell culture

The YUSIK and YUMAC human metastatic melanoma cell lines (Yale Skin Disease Research Core Center, New Haven, CT, USA) were engineered to express human full length Plexin B1 receptor using Lentiviral vectors as previously described (Stevens et al., 2010). YUSIK and YUMAC melanoma cells do not exhibit activation of c-Met receptor under basal conditions, but do respond to the c-Met ligand HGF by phosphorylation of the c-Met receptor (Stevens et al., 2010). The clone “PBA” (YUSIK; characterized in Stevens et al., 2010) and 1B2 (YUMAC; Figure S1) were used. Transductants were maintained in OptiMEM (Gibco, Carlsbad, CA, USA) with 5% fetal bovine serum (HyClone, Logan, UT, USA) and blasticidin (Invitrogen, Carlsbad, CA, USA) at 2 μg/ml.

Staining of cells for actin

105 cells were plated onto Lab-Tek Chamber Slides (Nalge Nunc International, Naperville IL, USA) and were allowed to attach for 24 h. Cells were placed in basal media 24 h prior to staining. Cells were rinsed with phosphate buffered saline (PBS) then fixed with 3.7% formalin in PBS for 10 min. Cells were permeabilized with 0.1% Triton-X/PBS for 3 min and then rinsed with PBS for 5 min. Slides were incubated with Fluoro-594-phalloidin as per manufacturer's instructions, and then rinsed with PBS and coverslipped.

Western blotting

Cells were lysed on ice using radioimmunoprecipitation assay buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl) with protease inhibitors (Boehringer Mannheim GmbH, Mannheim, Germany) and phosphatase inhibitors (GBiosciences, St. Louis, MO, USA). Lysates were resolved on SDS-PAGE and transferred to nitrocellulose paper. Immunoreactive proteins were visualized using SuperSignal West Pico Substrate (Pierce Chemical, Rockford, IL, USA).

FACS analysis

For analysis of β1-integrin surface expression, cells were trypsinized from the dish and the action of trypsin was stopped with soybean trypsin inhibitor. 106 cells were placed in PBS/0.1% bovine serum albumen and incubated with phycoerythrin-conjugated mouse monoclonal antibodies against β1-integrins (clone 4B7R, Abcam, Cambridge, MA, USA). Negative controls consisted of cells stained with phycoerythrin-conjugated mouse IgG (Sigma Co.).

Migration assay

Migration assays were performed using Chemicon QCM Chemotaxis 96-well Fluorometric Cell Migration Assay with 8 μm pore size (Millipore, Bedford, MA, USA). 105 cells were plated into the top well of the migration chamber in basal medium and incubated at 37°C for 24 h. The bottom wells contained basal media alone, or basal media with fibronectin in a range of doses. Cells were detached from the underside of the migration chamber plate with detachment solution and lysed and labeled with CyQuant GR Dye as described in the kit protocol. Fluorescence was measured at 480/520 nm with a Modulus Microplate Photometer (Turner Biosciences, Sunnyvale CA, USA). Background florescence (fluorescence of labeled media, in the absence of cells) was subtracted.

Invasion assay

Invasion assays were performed using CytoSelect 24-Well Cell Invasion Assay (basement membrane on 8 μ pore filters) from Cell Biolabs (San Diego, CA, USA). 5 × 104 cells were plated onto the top well of the invasion chamber in basal medium and incubated at 37°C for 24 h. Cells that invaded through the membrane were detached with dissociation buffer, lysed and quantified with CyQuant GR dye. Fluorescence was measured at 480/520 nm with a Modulus Microplate Photometer. Background florescence (fluorescence of labeled media, in the absence of cells) was subtracted.

Comparative real time PCR for pp125FAK

Total RNA from YUMAC and YUSIK cells was isolated using the RNeasy Mini Kit (QIAgen, Valencia, CA, USA) according to manufacturer's instructions. Reverse transcription was performed using 0.75 μg of total RNA with SuperScript II reverse transcriptase (Invitrogen). PCR was performed using iQ SYBR Green Supermix (BioRad Laboratories) on the Applied Biosystems ABI prism 7700 sequencedetection system (BioRad iCycler). Primers used for amplification of pp125FAK were: fwd: 5′-ATT GCT GCC TCG GAA TGT TCT -3′; rvs: 5′-GCT GAG GTA AAA CGT CGA AAA-3′. The conditions were: 94°C, 3 min (1 cycle); 94°C 45 sec, 57.8°C, 30 sec, 72°C, 60 sec (40 cycles). Primers used for amplification of β-actin were: fwd: 5′-CAC-GCACGATTTCCCGCTCGG-3′; rvs: 5′-CAGGCTGTGCTATCCTGTAC-3′. The conditions were 95°C, 3 min (1 cycle); 95°C 1 5 sec, 54.5°C, 30 sec, 72°C, 40 sec (40 cycles). Samples were analyzed in triplicate, and each analysis was performed four times. Fold change in mRNA between samples was calculated using the Pfaffl equation (Pfaffl, 2001) using β-actin as a reference gene. Efficiency of β-actin amplification was assumed to be 94.3%; efficiency of pp125FAK amplification was assumed to be 93.6%.

Statistical analysis

Differences between means were analyzed by two-tailed Student's t-test. A p value <0.05 was considered significant.

Supplementary Material

Supplemental Fig. 1

Figure S1. Expression of Plexin B1 in YUMAC melanoma clones.

Supplemental Fig. 2

Significance.

Loss of the semaphorin receptor Plexin B1 promotes late stages of melanoma progression. pp125FAK and Rho play a critical role in regulation of actin assembly, cell migration, and cell survival, and aberrant activation is associated with tumor progression. The present work is significant because it identifies pp125FAK and Rho as downstream targets of Plexin B1 in melanoma. We propose that Plexin B1 suppresses progression of melanoma through inhibitory effects on multiple targets, including c-Met, pp125FAK and Rho. Suppression of pp125FAK and Rho activity by Plexin B1 in melanoma could result in suppression of cell migration, angioinvasion, and anchorage-independent growth.

Acknowledgments

The work presented in this manuscript is supported by RO1AR45427-04 (GS).

Footnotes

Supporting information: Additional Supporting Information may be found in the online version of this article:

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

References

  1. Akasaka T, Van Leeuwen RL, Yoshinaga IG, Mihm MC, Jr, Byers HR. Focal adhesion kinase (p125FAK) expression correlates with motility of human melanoma cell lines. J Invest Dermatol. 1995;105:104–108. doi: 10.1111/1523-1747.ep12313396. [DOI] [PubMed] [Google Scholar]
  2. Argast GM, Croy CH, Couts KL, Zhang Z, Litman E, Chan DC, Ahn NG. Plexin B1 is repressed by oncogenic B-Raf signaling and functions as a tumor suppressor in melanoma cells. Oncogene. 2009;28:2697–2709. doi: 10.1038/onc.2009.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arnaout MA, Goodman SL, Xiong JP. Structure and mechanics of integrin-based cell adhesion. Curr Opin Cell Biol. 2007;19:495–507. doi: 10.1016/j.ceb.2007.08.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bagnard D. Plexins: making links to the cytoskeleton and reducing the GAPs in our knowledge. Trends Cell Biol. 2001;11:107. doi: 10.1016/s0962-8924(01)01931-6. [DOI] [PubMed] [Google Scholar]
  5. Barberis D, Casazza A, Sordella R, Corso S, Artigiani S, Settleman J, Comoglio PM, Tamagnone L. p190 Rho-GTPase activating protein associates with plexins and it is required for semaphorin signalling. J Cell Sci. 2005;118(Pt 20):4689–4700. doi: 10.1242/jcs.02590. [DOI] [PubMed] [Google Scholar]
  6. Barry ST, Flinn HM, Humphries MJ, Critchley DR, Ridley AJ. Requirement for Rho in integrin signalling. Cell Adhes Commun. 1997;4:387–398. doi: 10.3109/15419069709004456. [DOI] [PubMed] [Google Scholar]
  7. Castellani V, Rougon G. Control of semaphorin signaling. Curr Opin Neurobiol. 2002;12:532–541. doi: 10.1016/s0959-4388(02)00357-4. [DOI] [PubMed] [Google Scholar]
  8. Conrotto P, Corso S, Gamberini S, Comoglio PM, Giordano S. Interplay between scatter factor receptors and B plexins controls invasive growth. Oncogene. 2004;23:5131–5137. doi: 10.1038/sj.onc.1207650. [DOI] [PubMed] [Google Scholar]
  9. Dirisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet. 1996;14:457–460. doi: 10.1038/ng1296-457. [DOI] [PubMed] [Google Scholar]
  10. Fujisawa H. Discovery of semaphorin receptors, neuropilin and plexin, and their functions in neural development. J Neurobiol. 2004;59:24–33. doi: 10.1002/neu.10337. [DOI] [PubMed] [Google Scholar]
  11. Gawecka JE, Griffiths GS, Ek-Rylander B, Ramos JW, Matter ML. R-Ras regulates migration through an interaction with filamin A in melanoma cells. PLos One. 2010;23:e11269. doi: 10.1371/journal.pone.0011269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilmore AP. Anoikis. Cell Death & Differen. 2005;12(Suppl. 2):1473–1477. doi: 10.1038/sj.cdd.4401723. [DOI] [PubMed] [Google Scholar]
  13. Giordano S, Corso S, Conrotto P, Artigiani S, Gilestro G, Barberis D, Tamagnone L, Comoglio PM. The semaphorin 4D receptor controls invasive growth by coupling with Met. Nat Cell Biol. 2002;4:720–724. doi: 10.1038/ncb843. [DOI] [PubMed] [Google Scholar]
  14. Golubovskaya VM, Kweh FA, Cance WG. Focal adhesion kinase and cancer. Histol Histopathol. 2009;24:503–510. doi: 10.14670/HH-24.503. [DOI] [PubMed] [Google Scholar]
  15. Gomez Roman JJ, Garay GO, Saenz P, et al. Plexin B1 is downregulated in renal cell carcinomas and modulates cell growth. Transl Res. 2008;151:134–140. doi: 10.1016/j.trsl.2007.12.003. [DOI] [PubMed] [Google Scholar]
  16. Halaban R, Rubin JS, White W. met and HGF-SF in normal melanocytes and melanoma cells. Exs. 1993;65:329–339. [PubMed] [Google Scholar]
  17. Hall A, Paterson HF, Adamson P, Ridley AJ. Cellular responses regulated by rho-related small GTP-binding proteins. Philos Trans R Soc Lond B Biol Sci. 1993;340:267–271. doi: 10.1098/rstb.1993.0067. [DOI] [PubMed] [Google Scholar]
  18. Hanks SK, Ryzhova L, Shin NY, Brabek J. Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci. 2003;8:d982–d996. doi: 10.2741/1114. [DOI] [PubMed] [Google Scholar]
  19. Hao H, Naomoto Y, Bao X, et al. Focal adhesion kinase as potential target for cancer therapy (Review) Oncol Rep. 2009;22:973–979. doi: 10.3892/or_00000524. [DOI] [PubMed] [Google Scholar]
  20. Heasman SJ, Ridley AJ. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol. 2008;9:690–701. doi: 10.1038/nrm2476. [DOI] [PubMed] [Google Scholar]
  21. Hess AR, Postovit LM, Margaryan NV, Seftor EA, Schneider GB, Seftor RE, Nickoloff BJ, Hendrix MJ. Focal adhesion kinase promotes the aggressive melanoma phenotype. Cancer Res. 2005;65:9851–9860. doi: 10.1158/0008-5472.CAN-05-2172. [DOI] [PubMed] [Google Scholar]
  22. Hotchin NA, Hall A. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J Cell Biol. 1995;131:1857–1865. doi: 10.1083/jcb.131.6.1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Huggins CE, Domenighetti AA, Ritchie ME, Khalil N, Favaloro JM, Proietto J, Smyth GK, Pepe SV, Delbridge LM. Functional and metabolic remodelling in GLUT4-deficient hearts confers hyperresponsiveness to substratum intervention. J Mol Cell Cardio. 2008;44:270–280. doi: 10.1016/j.yjmcc.2007.11.020. [DOI] [PubMed] [Google Scholar]
  24. Kahana O, Micksche M, Witz IP, Yron I. The focal adhesion kinase (P125FAK) is constitutively active in human malignant melanoma. Oncogene. 2002;21:3969–3977. doi: 10.1038/sj.onc.1205472. [DOI] [PubMed] [Google Scholar]
  25. Kanner SB, Reynolds AB, Vines RR, Parsons JT. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A. 1990;87:3328–3332. doi: 10.1073/pnas.87.9.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Korostylev A, Worzfeld T, Deng S, et al. A functional role for semaphorin 4D/plexin B1 interactions in epithelial branching morphogenesis during organogenesis. Development. 2008;135:3333–3343. doi: 10.1242/dev.019760. [DOI] [PubMed] [Google Scholar]
  27. Lydolph MC, Morgan-Fisher M, Hoye AM, Couchman JR, Wewer UM, Yoneda A. Alpha9beta1 integrin in melanoma cells can signal different adhesion states for migration and anchorage. Exp Cell Res. 2009;315:3312–3324. doi: 10.1016/j.yexcr.2009.09.022. [DOI] [PubMed] [Google Scholar]
  28. Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. 2005;6:56–68. doi: 10.1038/nrm1549. [DOI] [PubMed] [Google Scholar]
  29. Moretti S, Procopio A, Boemi M, Catalano A. Neuronal semaphorins regulate a primary immune response. Curr Neurovasc Res. 2006;3:295–305. doi: 10.2174/156720206778792939. [DOI] [PubMed] [Google Scholar]
  30. Nakamura F, Kalb RG, Strittmatter SM. Molecular basis of semaphorin-mediated axon guidance. J Neurobiol. 2000;44:219–229. doi: 10.1002/1097-4695(200008)44:2<219::aid-neu11>3.0.co;2-w. [DOI] [PubMed] [Google Scholar]
  31. Narumiya S, Tanji M, Ishizaki T. Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion. Cancer Metastasis Rev. 2009;28:65–76. doi: 10.1007/s10555-008-9170-7. [DOI] [PubMed] [Google Scholar]
  32. Negishi M, Oinuma I, Katoh H. Plexins: axon guidance and signal transduction. Cell Mol Life Sci. 2005;62:1363–1371. doi: 10.1007/s00018-005-5018-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Neufeld G, Shraga-Heled N, Lange T, Guttmann-Raviv N, Herzog Y, Kessler O. Semaphorins in cancer. Front Biosci. 2005;10:751–760. doi: 10.2741/1569. [DOI] [PubMed] [Google Scholar]
  34. Oinuma I, Katoh H, Negishi M. Molecular dissection of the semaphorin 4D receptor plexin-B1-stimulated R-Ras GTPase-activating protein activity and neurite remodeling in hippocampal neurons. J Neurosci. 2004;24:11473–11480. doi: 10.1523/JNEUROSCI.3257-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oinuma I, Katoh H, Negishi M. Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity. J Cell Biol. 2006;173:601–613. doi: 10.1083/jcb.200508204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parsons JT, Martin KH, Slack JK, Taylor JM, Weed SA. Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement. Oncogene. 2000;19:5606–5613. doi: 10.1038/sj.onc.1203877. [DOI] [PubMed] [Google Scholar]
  37. Perrot V, Vazquez-Prado J, Gutkind JS. Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF. J Biol Chem. 2002;277:43115–43120. doi: 10.1074/jbc.M206005200. [DOI] [PubMed] [Google Scholar]
  38. Pfaffl MW. A new mathematical model for relative quantification in real time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Potiron VA, Roche J, Drabkin HA. Semaphorins and their receptors in lung cancer. Cancer Lett. 2009;273:1–14. doi: 10.1016/j.canlet.2008.05.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reddig PJ, Juliano RL. Clinging to life: cell to matrix adhesion and cell survival. Cancer Metastasis Rev. 2005;24:425–439. doi: 10.1007/s10555-005-5134-3. [DOI] [PubMed] [Google Scholar]
  41. Ridley AJ. The GTP-binding protein Rho. Int J Biochem Cell Biol. 1997;29:1225–1229. doi: 10.1016/s1357-2725(97)00052-6. [DOI] [PubMed] [Google Scholar]
  42. Ridley AJ. Rho family proteins: coordinating cell responses. Trends Cell Biol. 2001;11:471–477. doi: 10.1016/s0962-8924(01)02153-5. [DOI] [PubMed] [Google Scholar]
  43. Rody A, Holtrich U, Gaetje R, et al. Poor outcome in estrogen receptor-positive breast cancers predicted by loss of plexin B1. Clin Cancer Res. 2007;13:1115–1122. doi: 10.1158/1078-0432.CCR-06-2433. [DOI] [PubMed] [Google Scholar]
  44. Rody A, Karn T, Ruckhaberle E, et al. Loss of Plexin B1 is highly prognostic in low proliferating ER positive breast cancers–results of a large scale microarray analysis. Eur J Cancer. 2009;45:405–413. doi: 10.1016/j.ejca.2008.10.016. [DOI] [PubMed] [Google Scholar]
  45. Routhier A, Astuccio M, Lahey D, et al. Pharmacological inhibition of Rhokinase signaling with Y-27632 blocks melanoma tumor growth. Oncol Rep. 2010;23:861–867. [PubMed] [Google Scholar]
  46. Ruth MC, Xu Y, Maxwell IH, Ahn NG, Norris DA, Shellman YG. RhoC promotes human melanoma invasion in a PI3K/Akt-dependent pathway. J Invest Dermatol. 2006;126:862–868. doi: 10.1038/sj.jid.5700211. [DOI] [PubMed] [Google Scholar]
  47. Saito Y, Oinuma I, Fujimoto S, Negishi M. Plexin-B1 is a GTPase activating protein for M-Ras, remodelling dendrite morphology. EMBO Rep. 2009;10:614–621. doi: 10.1038/embor.2009.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sanz-Moreno V, Marshall CJ. Rho-GTPase signaling drives melanoma cell plasticity. Cell Cycle. 2009;8:1484–1487. doi: 10.4161/cc.8.10.8490. [DOI] [PubMed] [Google Scholar]
  49. Schena M, Shalon D, Heller R, Chai A, Brown PO, Davis RW. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc Natl Acad Sci USA. 1996;93:10614–10619. doi: 10.1073/pnas.93.20.10614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Scott G, Liang H. pp125FAK in human melanocytes and melanoma: expression and phosphorylation. Exp Cell Res. 1995;219:197–203. doi: 10.1006/excr.1995.1219. [DOI] [PubMed] [Google Scholar]
  51. Scott G, Ryan DH, Mccarthy JB. Molecular mechanisms of human melanocyte attachment to fibronectin. J Invest Dermatol. 1992;99:787–794. doi: 10.1111/1523-1747.ep12614749. [DOI] [PubMed] [Google Scholar]
  52. Stevens L, Mcclelland L, Fricke A, Williamson M, Kuo I, Scott G. Plexin B1 suppresses c-met in melanoma: a role for plexin B1 as a tumor-suppressor protein through regulation of c-met. J Invest Dermatol. 2010;30:1636–1645. doi: 10.1038/jid.2010.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Takaishi K, Sasaki T, Kato M, Yamochi W, Kuroda S, Nakamura T, Takeichi M, Takai Y. Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility. Oncogene. 1994;9:273–279. [PubMed] [Google Scholar]
  54. Takegahara N, Takamatsu H, Toyofuku T, et al. Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis. Nat Cell Biol. 2006;8:615–622. doi: 10.1038/ncb1416. [DOI] [PubMed] [Google Scholar]
  55. Tian J, Paquette-Straub C, Sage EH, Funk SE, Patel V, Galileo D, Mclane MA. Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin. Toxicon. 2007;50:448–452. doi: 10.1016/j.toxicon.2006.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Timar J, Tovari J, Szekeres K, Kagawa D, Honn KV. Key determinants of the invasion mechanism of melanoma. Role for a new signaling pathway Adv Exp Med Biol. 1997;407:303–310. doi: 10.1007/978-1-4899-1813-0_45. [DOI] [PubMed] [Google Scholar]
  57. To CT, Tsao MS. The roles of hepatocyte growth factor/scatter factor and met receptor in human cancers (Review) Oncol Rep. 1998;5:1013–1024. doi: 10.3892/or.5.5.1013. [DOI] [PubMed] [Google Scholar]
  58. Tomar A, Schlaepfer DD. Focal adhesion kinase: switching between GAPs and GEFs in the regulation of cell motility. Curr Opin Cell Biol. 2009;21:676–683. doi: 10.1016/j.ceb.2009.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vink J, Thomas L, Etoh T, Bruijn JA, Mihm MC, Jr, Gattoni-Celli S, Byers HR. Role of beta-1 integrins in organ specific adhesion of melanoma cells in vitro. Lab Invest. 1993;68:192–203. [PubMed] [Google Scholar]
  60. Wong OG, Nitkunan T, Oinuma I, et al. Plexin-B1 mutations in prostate cancer. Proc Natl Acad Sci U S A. 2007;104:19040–19045. doi: 10.1073/pnas.0702544104. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Fig. 1

Figure S1. Expression of Plexin B1 in YUMAC melanoma clones.

NIHMS464714-supplement-Supplemental_Fig__1.jpg (113.2KB, jpg)
Supplemental Fig. 2
NIHMS464714-supplement-Supplemental_Fig__2.jpg (39KB, jpg)

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