p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells

Biochemical and Biophysical Research Communications 392 (2010) 403–408

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p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells Katsuya Sakai a, Kiyomasa Oka a, Kunio Matsumoto b, Toshikazu Nakamura a,* a

Kringle Pharma Joint Research Division for Regenerative Drug Discovery, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan b Division of Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan

a r t i c l e

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Article history: Received 17 December 2009 Available online 13 January 2010 Keywords: Heparan sulfate proteoglycan Perlecan Endothelial cell p27

a b s t r a c t Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Perlecan is a large, secreted heparan sulfate proteoglycan that is a major component of basement membranes, interstitial matrix of cartilage, and bone stroma [1–3]. The core protein is composed of five distinct domains that show homology with the low-density lipoprotein receptor, epidermal growth factor, laminin, and neural-cell adhesion molecules [1–3]. The NH2 terminus of perlecan contains three glycosaminoglycan attachment sites. These glycosaminoglycan side chains, as well as domains of the core protein, interact with a wide range of biological molecules including extracellular matrix (ECM), growth factors, morphogens, and cell adhesion molecules [1–3], suggesting that perlecan integrates the diverse functions of these molecules. However, the physiological relevance of these interactions remains largely unknown. Among the known functions of perlecan, its role as a regulator of angiogenesis and wound healing via fibroblast growth fac-

Abbreviations: ECM, extracellular matrix; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial cell growth factor; CDK, cyclin-dependent kinase; BrdU, 5-bromo-20 -deoxyuridine; FBS, fetal bovine serum; HUVECs, human umbilical vein endothelial cells; siRNA, small interference RNA; FAK, focal adhesion kinase * Corresponding author. Fax: +81 6 6879 4130. E-mail address: [email protected] (T. Nakamura). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.01.035

tor (FGF) signaling has been most extensively studied. By participating in the high-affinity binding of basic fibroblast growth factor (bFGF) to FGF receptors, perlecan possesses angiogenic properties and promotes tumor angiogenesis [4]. Antisense blocking of perlecan expression inhibited the biological effects of bFGF in fibroblasts and melanoma cells [5] and of FGF-7 in colon calcinoma cells [6], and blocked tumor angiogenesis [6]. Genetically altered mice lacking the heparan sulfate side chain of perlecan showed impaired bFGF-induced corneal angiogenesis and reduced tumor angiogenesis and wound repair [7]. Thus, the pro-angiogenic effect of perlecan has been attributed to its function as a low-affinity co-receptor with members of the FGF family. Recently, we investigated the interaction between perlecan and NK4 [8], which is a fragment of hepatocyte growth factor and an angiogenesis inhibitor [9,10]. We found that the knockdown of perlecan in vascular endothelial cells inhibited cell proliferation with no interference with bFGF-induced signaling. The results of the present study show that perlecan-deficient endothelial cells have increased levels of the p27 protein, which is an inhibitor of cyclin-dependent kinase (CDK). In addition, p27 is predominantly localized in the nucleus of perlecan-deficient cells. Our results suggest that perlecan has pro-angiogenic effects via a mechanism that is distinct from its participation in bFGFdependent signaling.

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Materials and methods Materials and antibodies. bFGF and vascular endothelial growth factor (VEGF)-A165 were obtained from R&D Systems. The antibodies used in this study were as follows: anti-cyclin D1 (DCS6), anti-paxillin (349), anti-p27 (G173-524), and anti-5-bromo20 -deoxyuridine (BrdU, B44), BD Biosciences; anti-p16INK4 and anti-p21 (SX118), BD Pharmingen; anti-Cdk2 (D-12), anti-Cdk4 (C-22), anti-VEGF-R2 (C-1158), anti-phospho-tyrosine (PY99), and anti-Rb (C-15), Santa Cruz Biotechnology; anti-a-tubulin (B5-1-1), Sigma; anti-phospho-ERK1/2 (E10) and anti-phospho-Rb (pS780), Cell Signaling; anti-ERK1/2, Upstate Biotechnology; anti-phospho-focal adhesion kinase (FAK, pY397), Biosource; anti-laminin c1 (A5), Chemicon; anti-perlecan domain III (7B5), Zymed Laboratories; and, anti-heparan sulfate (F58-10E4), Seikagaku. Cell culture. Human umbilical vein endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs) were purchased from Cambrex Bio Science. HUVECs were cultured in MCDB131 medium supplemented with 5% fetal bovine serum (FBS), 2 mM L-glutamine, and 10 ng/ml bFGF. HPAECs and HPASMCs were cultured according to the manufacturer’s instructions. Supplemented growth factors were epidermal growth factor, bFGF, VEGF, and insulin-like growth factor-1 for HPAECs; epidermal growth factor, bFGF, and insulin for HPASMCs. Western blot and immunocytochemistry. Cells were lysed in 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 25 mM b-glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, and protease inhibitors (1 mM phenylmethylsufonyl fluoride, 1 lg/ml of aprotinin, pepstatin A, and leupeptin) on ice. Determination of protein concentration, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), electroblotting, and reaction with antibodies were performed as described previously [8]. For immunoprecipitation, an aliquot of cell lysate containing 400 lg of protein was pre-treated with protein A–Sepharose, incubated with 2 lg anti-VEGF-R2 antibody at 4 °C overnight, and precipitated with protein A–Sepharose at 4 °C for 2 h. For immunofluorescent staining, cells on a coverglass were fixed and stained with antibodies as described previously [8]. Cells were visualized using a laser-scanning confocal microscope (LSM5 PASCAL; Carl Zeiss). Images were adjusted for brightness and contrast, and cropped with Photoshop 6.0 (Adobe) software. The fluorescence intensity of phosho-FAK and paxillin at cell edges was analyzed using WinROOF software (Mitani). Small interference RNA (siRNA). siRNA oligonucleotides were obtained from Nippon EGT. The siRNA sequences for perlecan were as follows: si-P3105 sense, 50 -CAUCAUCCUAGAGCACCAUtt-30 and anti-sense, 30 -ttGUAGUAGGAUCUCGUGGUA-50 ; si-P6159 sense, 50 GAUUGAGUCCUCAUCGCCUtt-30 and anti-sense, 30 -ttCUAACUCAG GAGUAGCGGA-50 ; and, si-P9461 sense, 50 -GUUGGAGCAGCGGAC AUAUtt-30 and anti-sense, 30 -ttCAACCUCGUCGCCUGUAUA-50 . Random siRNA was used as a control. Cells were transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and then allowed to recover for 16 h in complete medium before each assay. RNA extraction, cDNA preparation, and real-time analysis of reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted using the TRIZOL reagent (Invitrogen). First-strand cDNAs were synthesized using SuperScript III Reverse Transcriptase (Invitrogen) with a random hexamer. The primer sequences were as follows: human perlecan (forward primer, 50 -ACAGTGCAACAAGT GCAAGG-30 and reverse primer, 50 -CTGAAGTGACCAGGCTCCTC-30 ); human p27 (forward primer, 50 -AGATGTCAAACGTGCGAGTG-30 and reverse primer, 50 -TCTCTGCAGTGCTTCTCCAA-30 ); human GAP-

DH (forward primer, 50 -GAGTCAACGGATTTGGTCGT-30 and reverse primer, 50 -GACAAGCTTCCCGTTCTCAG-30 ). The PRISM 7000 realtime PCR system (Applied Biosystems) and Power SYBER Green PCR Master Mix (Applied Biosystems) were used for the amplification and online detection. Experimental samples were matched to the standard curve generated by amplifying serially diluted products using the same PCR protocol. Cell adhesion and migration assay. Migration of HUVECs was assayed using Transwell systems (Costar, 8 lm pore, 6.5 mm diameter). The lower membranes of the inserts were pre-coated with indicated concentrations of fibronectin, 3 lg/ml of collagen type I, laminin, or vitronectin, followed by blocking with 1% bovine serum albumin. MCDB131 medium containing 0.2% bovine serum albumin (300 ll) and HUVECs (1  104 cells/insert) transfected with siRNA were added to the upper insert of a Transwell. In the bottom chamber, 800 ll of MCDB131 medium containing 0.2% bovine serum albumin was added. The cells were cultured for either 1.5 h (fibronectin) or 2.5 h (for others), and were then fixed with 4% paraformaldehyde in PBS. Cells that attached to the top of the membrane were thoroughly scraped off and cells that attached to the bottom the membrane were stained with 0.4% crystal violet. The number of cells that migrated to the undersurface of the filter was determined by counting cells in five randomly selected microscopic fields (200) in each well. Results Perlecan knockdown impaired DNA synthesis in human endothelial cells. Perlecan expression was evaluated in HUVECs transfected with control siRNA or siRNA for perlecan (three different sequences) by immunocytochemistry using anti-perlecan antibody (Fig. 1A) and real-time RT-PCR (Fig. 1B). In both assays, inhibition of perlecan expression was greatest in HUVECs treated with siP9461, while expression was moderately reduced with si-P3105 and si-P6159. To test the specificity of siRNA-mediated perlecan knockdown, HUVECs treated with control siRNA or si-P9461 were triple-immunostained for perlecan, laminin c1 (LN), and heparan sulfate proteoglycans (HS) (Fig. 1C). Perlecan expression was significantly diminished by si-P9461, whereas expression of other heparan sulfate proteoglycans and laminin c1 was unchanged by si-P9461, indicating that si-P9461-treatment selectively inhibited perlecan expression. Next, we evaluated DNA synthesis in HUVECs transfected with control siRNA or siRNA for perlecan in the presence or absence of bFGF (Fig. 1D) or VEGF (Fig. 1E). HUVECs treated with si-P9461 showed almost complete impairment of bFGF- or VEGF-induced DNA synthesis. The impairment in bFGF-dependent DNA synthesis by si-P3105, si-P6159, or si-P9641 was correlated with the degree of perlecan knockdown (Fig. 1D). Importantly, even in the absence of bFGF or VEGF, perlecan knockdown decreased DNA synthesis, suggesting that the role of perlecan in endothelial cell growth might be independent of growth factor signaling. To elucidate the role of perlecan in DNA synthesis in different cell types, we used siRNA-mediated perlecan knockdown in human pulmonary artery endothelial cells and pulmonary artery smooth muscle cells. Transfection of these cells with si-P9461 resulted in effective perlecan mRNA knockdown compared with the cells transfected with control siRNA (Fig. 1B). Knockdown of perlecan expression decreased DNA synthesis in pulmonary artery endothelial cells (Fig. 1F). This result provides additional evidence that perlecan is required for endothelial cell growth. By contrast, perlecan knockdown enhanced DNA synthesis in smooth muscle cells (Fig. 1G). This is consistent with previous reports that perlecan suppressed proliferation of rat smooth muscle cells [1,11,12].

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Fig. 1. Inhibition of DNA synthesis by siRNA-mediated knockdown of perlecan in human endothelial cells. (A) Immunostaining of pericellular and intracellular perlecan in human umbical vein endothelial cells (HUVECs) transfected with either control siRNA or siRNA for perlecan (three different sequences). Scale bar, 20 lm. (B) Changes in perlecan mRNA resulting from siRNA treatment. Perlecan mRNA in HUVECs, human pulmonary artery artery endothelial cells (HPAECs), and human pulmonary artery smooth muscle cells (HPASMCs) transfected with either control siRNA or siRNA for perlecan was quantified using real-time RT-PCR. Relative perlecan mRNA levels normalized to GAPDH mRNA are presented as means ± SD. (C) Distribution of perlecan, laminin c1 (LN), and heparan sulfate proteoglycans (HS) in HUVECs transfected with either control siRNA (si-C) or siRNA for perlecan (si-P9461). Scale bar, 20 lm. (D–G) Effects of perlecan knockdown on DNA synthesis in HUVECs (D,E), human pulmonary artery endothelial cells (F), and human pulmonary artery smooth muscle cells (G). Cells treated with either control siRNA or siRNA for perlecan were cultured in serum- and growth factor-free medium for 24 h. The cells were replated and cultured in medium supplemented with either serum (basal medium) or with serum and 20 ng/ml of bFGF (D,F) or VEGF (E) or growth factors described in Materials and methods (F,G) (growth medium) for another 24 h. DNA synthesis in each condition was determined by BrdU incorporation.

These results suggest that the effect of perlecan on DNA synthesis is cell-type specific. bFGF-induced signaling is not altered in perlecan-deficient HUVECs. To determine if the function of perlecan as a co-receptor for hepa-

rin-binding growth factors is responsible for the impaired DNA synthesis caused by the perlecan knockdown in endothelial cells, we analyzed bFGF-induced phosphorylation of ERK (Fig. 2A) and cyclin D1 expression (Fig. 2B) by Western blotting. Compared with

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Blot: VEGF-R2 IP: VEGF-R2 Fig. 2. Changes in growth factor-induced cellular signaling caused by lack of perlecan expression in HUVECs. (A,B) bFGF-induced changes in ERK phosphorylation (A) and expression levels of regulatory proteins involved in G1 checkpoint functions (B). (C) VEGF-induced changes in phosphorylation of VEGF-R2. HUVECs treated with either control siRNA or siRNA for perlecan (si-P9461) were cultured in serum- and growth factor-free medium for 24 h, and were then cultured in medium supplemented with or without 20 ng/ml of bFGF or VEGF for the indicated time periods in serum-free (A,C) or serum-supplemented (B) media. Total cell lysates were subjected to Western blotting (A,B). Tyrosine phosphorylation of VEGF-R2 was determined by immunoprecipitation of VEGF-R2 and Western blotting using an anti-phosphotyrosine antibody (C).

si-control-treated HUVECs, bFGF-induced ERK phosphorylation was unchanged in si-P9461-treated HUVECs (Fig. 2A). The bFGF-induced increase in cyclin D1 expression in si-P9461-treated HUVECs was nearly comparable to that observed in control cells (Fig. 2B). These results might be explained by functional compensation by other heparan sulfate proteoglycans [13,14]. Under the same knockdown conditions, VEGF-induced phosphorylation of VEGFR2 was significantly reduced in si-P9461-treated HUVECs (Fig. 2C). This is consistent with a previous report that perlecan plays a significant role in the regulation of the VEGF-VEGF-R2 axis both in zebrafish and in HUVECs [15]. Thus, in HUVECs, while perlecan is indispensable for VEGF signaling, it is dispensable for bFGF signaling. The impaired bFGF-induced DNA synthesis in perlecandeficient endothelial cells cannot be explained by the function of perlecan as a co-receptor with bFGF. Increased protein levels and nuclear localization of p27 by perlecan knockdown. Consistent with the defect in DNA synthesis, Rb phosphorylation after bFGF stimulation was significantly inhibited in siP9461-treated HUVECs (Fig. 2B), while the bFGF-induced increase in cyclin D1 expression and protein levels of Cdk2 and Cdk4 in

si-P9461-treated HUVECs was mostly comparable to that in control cells (Fig. 2B), which suggests the involvement of CDK inhibitors. Levels of the p16 and p21 proteins in si-P9461- and sicontrol-treated HUVECs were similar (Fig. 3A). By contrast, protein levels of p27 were significantly increased in si-P9461-treated HUVECs compared with si-control-treated cells (Fig. 3A). Although bFGF stimulation decreased p27 in si-P9461-treated cells, the level of p27 remained significantly greater than that in si-control-treated HUVECs without bFGF stimulation (Fig. 3A). These results indicate that perlecan knockdown in endothelial cells increases basal p27 protein expression, perhaps without affecting bFGF-induced p27 degradation. To determine if the increase in p27 protein that resulted from perlecan knockdown was accompanied by up-regulation of transcription, we quantified p27 mRNA abundance in si-P9461- or sicontrol-treated HUVECs with or without bFGF stimulation (Fig. 3B). p27 mRNA level was not significantly different between si-P9461- and si-control-treated HUVECs, regardless of bFGF stimulation. Next, we used immunocytochemistry to examine changes in the subcellular localization of p27 in si-P9461- or si-control-treated HUVECs (Fig. 3C). p27 was mostly localized in the nuclei of HUVECs that had been subjected to serum starvation for 24 h (data not shown). Eight hours after addition of serum to HUVECs treated with si-control, p27 was localized in the cytoplasm, regardless of bFGF stimulation. In si-P9461-treated cells, p27 was predominantly localized in the nucleus 8 h after serum addition. These results suggest that predominant nuclear localization of p27 participates in the impairment of DNA synthesis in perlecan-deficient endothelial cells. Static adhesion and decreased locomotion by perlecan knockdown. Because a loss of cellular adhesiveness is a primary cause of p27mediated growth arrest [16–18], the adhesive characteristic of cells was analyzed by measurement of the distribution and intensity of phosphorylated FAK and paxillin. Compared with control cells, HUVECs treated with si-P9461 exhibited enhanced phosphorylation of FAK along the cell edges (Fig. 4A and B). This result suggests more static interactions between the cells and substratum due to perlecan deficiency. Perlecan knockdown reproducibly resulted in continuous localization of phosphorylated FAK along the cell edges, even when HUVECs were cultured on different types of ECMs, including fibronectin, vitronectin, laminin, and type I collagen (Fig. 4C). Migration of endothelial cells is a critical step in angiogenesis, and cellular adhesion to ECM is regulated in a dynamic manner in locomoting cells. Therefore, we analyzed changes in HUVEC migration after perlecan knockdown (Fig. 4D and E). The number of cells that migrated to fibronectin, type I collagen, laminin, or vitronectin was decreased by perlecan knockdown. These results suggested that perlecan deficiency in vascular endothelial cells resulted in static and enhanced adhesion to ECM, thereby reducing cellular locomotion.

Discussion In addition to the well-known function of perlecan as a coreceptor for heparin-binding growth factors such as bFGF and VEGF [1–7,15], several studies have suggested that parlecan has a variety of functions. Perlecan inhibits smooth muscle cell proliferation via reduced FAK signaling and increased PTEN activity [11,12]. Perlecan is essential for survival of keratinocytes and epidermal formation, without affecting FGF-7-induced ERK activation and proliferation of keratinocytes [19]. We observed that knock down of perlecan expression in endothelial cells resulted in growth arrest without substantial interference with bFGF-induced ERK

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Fig. 3. Changes in p27 protein levels and nuclear localization caused by perlecan knockdown in HUVECs. (A) bFGF-induced changes in CDK inhibitors. (B) Changes in p27 mRNA levels. (C) Changes in subcellular localization of p27. HUVECs treated with either control siRNA or siRNA for perlecan (si-P9461) were cultured in serum- and growth factor-free medium for 24 h, and were then cultured in medium supplemented with serum with or without 20 ng/ml of bFGF for an appropriate duration. In (A), total cell lysates after a 20 h-culture were subjected to Western blotting. In (B), total RNA was extracted after the indicated culture periods and subjected to real-time RT-PCR. Relative perlecan mRNA levels normalized to GAPDH mRNA are presented as means ± SD. In (C), cells that had been cultured for 8 h were fixed and immunostained for p27. Nuclei were stained with To-pro3. Scale bar, 100 lm.

activation, cyclin D1 expression, or p27 degradation. We found that the increased protein level and nuclear localization of p27, and enhanced activation of FAK at the cell edge, were associated with both growth arrest and decreased cell motility in perlecan-deficient endothelial cells. Our results suggest regulation of angiogenesis by perlecan through a mechanism that involves p27 and FAK regulation, which differs from the function of perlecan as a coreceptor for heparin-binding growth factors. Because syndecan-4 in fibroblasts and syndecan-2 in colon cancer cells participate in cell adhesion to ECM, the lack of these proteoglycans is associated with impaired cell adhesion to ECM, thereby causes growth arrest through CDK inhibitors [20,21]. By contrast, perlecan deficiency in vascular endothelial cells did not impair cellular adhesion to ECM. Rather, perlecan deficiency caused a static interaction between the cells and ECM, which was associated with increased protein level and nuclear localization of p27 and growth arrest. Clearly, the regulatory function of perlecan in both anchoring of endothelial cells to ECM and in endothelial cell proliferation differs from the function of syndecans. Based on the cell-surface localization of perlecan, lack of perlecan in endothelial cells seems to reduce the dynamic interaction between the cells and ECM, which might lead to up-regulation of p27 and nuclear localization. A loss of perlecan could affect signaling from integrin, because perlecan binds to integrin

b1 and b3 [22], and the C-terminal fragment of perlecan endorepellin has angiostatic effects on endothelial cells via integrin a2b1 signaling [2,23]. Another possibility is that cell-surface perlecan might be required for the association of extracellular serum factor(s) that are capable of regulating subcellular localization of p27, because translocation of p27 is regulated by the presence of serum in endothelial cell cultures. Lipoproteins are possible serum factors that might regulate p27 translocation because they are major components of plasma and influence cell proliferation [24,25], while perlecan has a module domain of the LDL receptor [1–3]. In summary, based on perlecan knockdown in vascular endothelial cells, we found that perlecan has two functions: (1) perlecan plays a role in the dynamic interaction between cells and ECM, thereby regulating cell migration; and, (2) perlecan regulates cell growth by altering expression and subcellular localization of p27. These results suggest that perlecan has proangiogenic effects via a mechanism that differs from its wellcharacterized function as a co-receptor for heparin-binding growth factors. Elucidation of the mechanism by which perlecan regulates the cell–ECM interaction and p27 subcellular localization in endothelial cells will improve our understanding of the physiological and biological functions of perlecan in vascular regulation.

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Fig. 4. Changes in adhesiveness and migration of HUVECs caused by perlecan knockdown. (A) Distribution of phosphorylated FAK and paxillin. HUVECs treated with either control siRNA or siRNA for perlecan (si-P9461) were replated on coverglasses with medium containing serum, incubated for 3 h, fixed, and stained with anti-FAK phosphorylated at Y397 (pFAK, red) and anti-paxillin (green) antibodies. Scale bar, 100 lm. (B) Fluorescence intensity of pFAK and paxillin at cell edges. Fluorescence intensity on the line along with cell edges from a to b, or c to d, as indicated in (A), was analyzed. (C) Distribution of phosphorylated FAK and paxillin in HUVECs cultured on different types of ECM substrates. HUVECs transfected with either control siRNA or siRNA for perlecan (si-P9461) were replated on coverglasses coated with fibronectin (FN), collagen type I (COL), laminin (LN), or vitronectin (VN) in serum-free medium. The cells were cultured for 3 h, fixed, and stained for pFAK (red) and paxillin (green). Scale bar, 100 lm. (D and E) Effects of perlecan knockdown on HUVEC migration. Migration of HUVECs was assayed using Transwell chambers. The bottom of the membranes of the inserts were pre-coated with fibronectin (D), collagen type I, laminin, or vitronectin (E).

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