RNA Interference-mediated Silencing of the S100A10 Gene Attenuates Plasmin Generation and Invasiveness of Colo 222 Colorectal Cancer Cells
2004; Elsevier BV; Volume: 279; Issue: 3 Linguagem: Inglês
10.1074/jbc.m310357200
ISSN1083-351X
AutoresLibo Zhang, Darin K. Fogg, David M. Waisman,
Tópico(s)Cell Adhesion Molecules Research
ResumoS100A10 is a key plasminogen receptor of the extracellular cell surface that is overexpressed in many cancer cells. Typically, S100A10 is thought to be anchored to the plasma membrane via the phospholipid-binding sites of its binding partner, annexin A2. Here, using the potent and highly sequence-specific mechanism of RNA interference (RNAi), we have stably silenced the expression of the S100A10 gene in colorectal (CCL-222) cancer cells. We show that siRNA expression mediated by the pSUPER vector causes efficient, stable, and specific down-regulation of S100A10 gene expression. The siRNA-mediated down-regulation of S100A10 gene expression resulted in a major decrease in the appearance of extracellular S100A10 protein and correlated with a 45% loss of plasminogen binding, a 65% loss in cellular plasmin generation and a complete loss in plasminogen-dependent cellular invasiveness. We also observed that the CCL-222 cells do not express annexin A2 on their extracellular surface. Thus, the data show that annexin A2 is not required by S100A10 for its association with the plasma membrane, for its colocalization with uPAR, or for its binding and activation of plasminogen. S100A10 is a key plasminogen receptor of the extracellular cell surface that is overexpressed in many cancer cells. Typically, S100A10 is thought to be anchored to the plasma membrane via the phospholipid-binding sites of its binding partner, annexin A2. Here, using the potent and highly sequence-specific mechanism of RNA interference (RNAi), we have stably silenced the expression of the S100A10 gene in colorectal (CCL-222) cancer cells. We show that siRNA expression mediated by the pSUPER vector causes efficient, stable, and specific down-regulation of S100A10 gene expression. The siRNA-mediated down-regulation of S100A10 gene expression resulted in a major decrease in the appearance of extracellular S100A10 protein and correlated with a 45% loss of plasminogen binding, a 65% loss in cellular plasmin generation and a complete loss in plasminogen-dependent cellular invasiveness. We also observed that the CCL-222 cells do not express annexin A2 on their extracellular surface. Thus, the data show that annexin A2 is not required by S100A10 for its association with the plasma membrane, for its colocalization with uPAR, or for its binding and activation of plasminogen. Double-stranded RNA (dsRNA) 1The abbreviations used are: dsRNA, double strand RNA; BSA, bovine serum albumin; AIIt, annexin A2-S100A10 heterotetramer; p11, S100A10 subunit of annexin A2-S100A10 heterotetramer; RNAi, RNA interference; siRNA, small interfering RNA; ϵ-ACA, ϵ-aminocaproic acid; nt, nucleotide(s); tPA, tissue plasminogen activator; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; FBS, fetal bovine serum; mAb, monoclonal antibody; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; PFA, paraformaldehyde; RT, reverse transcription.-dependent post-transcriptional gene silencing, or RNA interference (RNAi), refers to the mechanism of sequence-specific, post-transcriptional gene silencing initiated by dsRNA homologous to the gene being suppressed. RNAi was originally described in Caenorhabditis elegans (1.Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (12141) Google Scholar, 2.Timmons L. Fire A. 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It was shown that during RNAi processing, long dsRNA was first degraded into 19–23 nt siRNA and then recruited into an RNA-induced silencing complex to degrade corresponding mRNA. Mechanistically, dsRNAs are processed to siRNA by Dicer, a cellular ribonuclease III, which generates duplexes of about 21 nt with 3′-overhangs (14.Hammond S.M. Bernstein E. Beach D. Hannon G.J. Nature. 2000; 404: 293-296Crossref PubMed Scopus (2453) Google Scholar, 15.Zamore P.D. Tuschl T. Sharp P.A. Bartel D.P. Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google Scholar). In mammalian cells siRNA molecules are capable of specifically silencing gene expression without cytotoxic effects. Presumably, these siRNA avoid provoking the PKR response by mimicking the products of the Dicer enzyme. Thus, siRNAs have become a novel and potent alternative to other genetic tools such as antisense oligonucleotides to probe for gene function. Typically the gene silencing produced by siRNA effects are short-lived, which severely limits its application to cellular systems. An alternative strategy uses the endogenous expression of siRNAs by various polymerase III promoter expression cassettes that allow transcription of functional siRNAs or their precursors. Agami's group reported the use of a vector system, named pSUPER that directs the synthesis of siRNA in mammalian cells (16.Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3979) Google Scholar). They demonstrated that the expression of siRNA by this vector resulted in the efficient and specific down-regulation of gene expression. Most importantly, this vector was used to establish the stable repression of gene expression. S100A10, a member of the S100 family of Ca2+-binding proteins, is a dimeric protein composed of two 11-kDa subunits (reviewed in Ref. 17.Donato R. Int. J. Biochem. Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1356) Google Scholar). The protein is cytosolic when present as a dimer. Typically, S100A10 is found in most cells bound to its annexin A2 ligand as the heterotetrameric (S100A10)2-(annexin A2)2 complex, AIIt. The formation of AIIt results in the translocation of S100A10 to the plasma membrane (reviewed in Refs. 18.Choi K.S. Fogg D.K. Fitzpatrick S.L. Waisman D.M. Bandorowicz-Pikula J. Annexins: Biological Importance and Annexin-related Pathologies. Landes Bioscience, Georgetown, TX2003: 218-233Crossref Google Scholar, 19.Filipenko N.R. Waisman D.M. Bandorowicz-Pikula J. Annexins: Biological Importance and Annexin-related Pathologies. Landes Bioscience, Georgetown, TX2003: 127-156Crossref Google Scholar, 20.Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1682) Google Scholar, 21.Donato R. Russo-Marie F. Cell Calcium. 1999; 26: 85-89Crossref PubMed Scopus (25) Google Scholar, 22.Seaton B.A. Dedman J.R. Biometals. 1998; 11: 399-404Crossref PubMed Scopus (54) Google Scholar). S100A10 has been shown to regulate plasma membrane ion channels (23.Girard C. Tinel N. Terrenoire C. Romey G. Lazdunski M. Borsotto M. EMBO J. 2002; 21: 4439-4448Crossref PubMed Scopus (137) Google Scholar, 24.Okuse K. Malik-Hall M. Baker M.D. Poon W.Y. Kong H. Chao M.V. Wood J.N. Nature. 2002; 417: 653-656Crossref PubMed Scopus (237) Google Scholar) as well as cytosolic phospholipase A2 (25.Wu T. Angus C.W. Yao X.L. Logun C. Shelhamer J.H. J. Biol. Chem. 1997; 272: 17145-17153Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In addition to an intracellular distribution, it has also been established that the heterotetrameric form of S100A10 is present on the extracellular surface of many cells (26.Yeatman T.J. Updyke T.V. Kaetzel M.A. Dedman J.R. Nicolson G.L. Clin. Exp. Metastasis. 1993; 11: 37-44Crossref PubMed Scopus (94) Google Scholar, 27.Tressler R.J. Updyke T.V. Yeatman T. Nicolson G.L. 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Biochemistry. 1998; 37: 16958-16966Crossref PubMed Scopus (117) Google Scholar, 34.Choi K.S. Fogg D.K. Yoon C.S. Waisman D.M. FASEB J. 2003; 17: 235-246Crossref PubMed Scopus (62) Google Scholar). The penultimate and ultimate carboxyl-terminal lysines of this subunit bind tPA and plasminogen (35.MacLeod T.J. Kwon M. Filipenko N.R. Waisman D.M. J. Biol. Chem. 2003; 278: 25577-25584Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and regulate the stimulation of tPA-dependent plasminogen activation (36.Fogg D.K. Bridges D.E. Cheung K.T. Kassam G. Filipenko N.R. Choi K.S. Fitzpatrick S.L. Nesheim M. Waisman D.M. Biochemistry. 2002; 41: 4953-4961Crossref PubMed Scopus (34) Google Scholar). In the present report we have used a modification of the pSUPER system, the selectable pSUPER.retro.circular.stuffer (OligoEngine) to stably suppress the expression of the S100A10 gene. CCL-22 colorectal cells were transfected with the pSUPER-S100A10 or pSUPER-Control vector, and transfectants were selected with puromycin. Resistant cells were cloned, and several stable lines were established. When analyzed after 4 months, we found that pSUPER-S100A10-transfected clones showed a significant reduction in S100A10 compared with the pSUPER-Control clones. In contrast, a similar strategy utilizing full-length antisense to S100A10 (pLIN-S100A10) failed to significantly lower S100A10 levels in the colorectal cells. The loss of S100A10 from the surface of the pSUPER-S100A10-transfected CCL-222 cells resulted in decreased plasmin production and a loss in cellular invasiveness. Thus we report for the first time the use of a stable siRNA system to study the function of the S100A10 gene. Materials—Human colorectal adenocarcinoma cell line CCL-222 cells were purchased from the American Type Culture Collection. The carcinoma cells were maintained according to the provider's instruction. The cells were cultured in RPMI-160 supplemented with 10% fetal bovine serum (FBS, Invitrogen). Recombinant S100A10 was expressed in Escherichia coli and purified as described previously (37.Kang H.M. Kassam G. Jarvis S.E. Fitzpatrick S.L. Waisman D.M. Biochemistry. 1997; 36: 2041-2050Crossref PubMed Scopus (41) Google Scholar). Glu-plasminogen and monoclonal anti-plasminogen antibody were purchased from American Diagnostica. Monoclonal anti-S100A10 antibody (BD Transduction Laboratories), polyclonal anti-annexin A2 (Santa Cruz Biotechnology) antibody, and goat polyclonal anti-uPAR antibody (Chemicon) were used for immunofluorescence. Rabbit anti-uPAR antibody and anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies was purchased from Santa Cruz Biotechnology. The monoclonal anti-annexin A2 antibody used in Western blots was from BD Transduction Laboratories. Anti-human α-tubulin monoclonal antibody was purchased from Oncogene Science. Small Interference RNA—The mammalian expression vector, pSU-PER.retro.circular.stuffer (OligoEngine) was used for expression of siRNA in CCL-222 cells. The gene-specific insert specifies a 19-nucleotide sequence corresponding to nucleotides 199–217 downstream of the transcription start site (gtgggcttccagagcttct) of S100A10, which is separated by a 9-nucleotide non-complementary spacer (tctcttgaa) from the reverse complement of the same 19-nucleotide sequence. This vector was referred to as pSUPER-S100A10. A control vector (pSUPER-Control) was constructed using a 19-nucleotide sequence (gcgcgctttgtaggattcg) with no significant homology to any mammalian gene sequence and therefore serves as a non-silencing control (OligoEngine). These sequences were inserted into the pSUPER.retro.circular.stuffer backbone after digestion with BglII and HindIII and transformed into BL21-A1 One Shot™ supercompetent cells (Invitrogen) according to the manufacturer's instructions. Several clones were obtained, and the vectors were amplified. To verify the insertion of the S100A10 sequence into the pSUPER backbone, the purified vector was digested with BstI (New England Biolabs). The S100A10 sequence chosen contains a restriction site for BstI; therefore, successful ligation of the sequence results in a restriction pattern distinct from that of unligated vector or vector ligated with the control sequence (data not shown). Transfection—CCL-222 cells were plated onto 6-well plates at 200,000 cells per well. After 24 h, cells were transfected with 1 μg of RNAi plasmid hybrids using LipofectAMINE 2000 reagent (Invitrogen, Rockville, MD) according to the manufacturer's instructions. 72 h after transfection, cells were processed for immunofluorescence analysis to evaluate S100A10 expression. Stable transfected cell lines were selected with 0.5 μg/ml puromycin (Sigma, St. Louis, MO); clonal cell lines were selected by S100A10 protein expression by Western blotting. Western Blotting—Total cell lysates were prepared in radioimmune precipitation assay lysis buffer (150 mm NaCl, 1% Nonidet P-40, 50 mm Tris-HCl, 0.1% SDS, 5 mm EDTA, and 20 mm NaF) supplemented with 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. Lysates were cleared by centrifugation at 14,000 × g for 20 min at 4 °C and analyzed by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with a 5% skim milk solution, the membrane was incubated with 0.25 μg/ml of an anti-human S100A10 mAb, 0.25 μg/ml of an anti-human Annexin II mAb, or 1 μg/ml of an anti-human α-tubulin mAb. These mAbs were detected with 0.2 μg/ml horseradish peroxidase-conjugated goat anti-mouse IgG and developed using a Super Signal detection kit (Pierce, Rockford, IL). Immunofluorescence Microscopy—Cells were cultured on coverslips until 80–90% confluence. Cells were then washed with ice-cold DPBS and fixed with 4% PFA (paraformaldehyde) at 4 °C. Alternatively, cells were permeabilized by fixation with ice-cold 100% methanol. After blocking with 1% BSA in PBS at 4 °C for 1 h, primary antibodies (monoclonal anti-S100A10 antibody and goat polyclonal anti-annexin A2 antibody) were applied to the cells (1 μg/ml in PBS at 4 °C) for 1 h. Fluorophore-labeled second antibody (Cy3-conjugated rabbit anti-mouse and Alexa-conjugated donkey anti-goat) was then applied at 4 °C for 1 h. After thoroughly washing with PBS, coverslips were mounted in a solution of Prolong AntiFade (Molecular Probes) and visualized using a Zeiss Axioskop microscope or confocal microscope. To differentiate non-specific binding of antibodies, isotype-matched, control mouse and goat antibodies were applied to cells, and incubated under the same conditions. RT-PCR—Total RNA was extracted by using the RNeasy Mini kit (Qiagen). Purified RNA was reverse-transcribed using the One-Step RT-PCR system (Qiagen) according to the manufacturer's protocol in 20 μl of final volume. Subsequently, 2 μl of cDNA was PCR-amplified using platinum TaqDNA polymerase (Invitrogen) for 22 cycles using S100A10-, annexin A2-, or glyceraldehyde-3-phosphate dehydrogenase cDNA-specific primers. Plasminogen Binding Assay—Recombinant human Glu-plasminogen (American Diagnostica) was radioiodinated as described previously (34.Choi K.S. Fogg D.K. Yoon C.S. Waisman D.M. FASEB J. 2003; 17: 235-246Crossref PubMed Scopus (62) Google Scholar). Plasminogen (10 μm) was incubated for 3 min at room temperature with 37 MBq (1 mCi) of Na125I and three IODO-BEADs (Pierce) in PBS. Free Na125I and protein were separated using a PD-10 column (Sephadex G-25, Amersham Biosciences, Uppsala, Sweden), equilibrated, and eluted with PBS. The specific activity of the protein preparations ranged from 1000 to 2000 cpm/pmol of protein. Confluent cells in 24-well plates were rinsed with ice-cold DPBS and incubated with 50 nm radioiodinated plasminogen and 0.45 μm cold plasminogen in the presence or absence of 10 mm ϵ-ACA at 4 °C for 1 h. The radioactivity of the cells was determined after washing the cells with ice-cold DPBS three times. Plasminogen Activation Assay—Both transfected and parental CCL-222 cells were seeded in 24-well culture plates at a density of 5 × 106 cells/ml. After incubation in RPMI 1640 with 10% bovine serum albumin for 6 h, cells were rinsed two times with PBS (pH 7.4), and culture media was replaced with fresh phenol-red- and serum-free RPMI-1604 media. Purified Glu-plasminogen (American Diagnostica) was added at a final concentration of 0.5 μm. Conditioned media was collected and cleared by centrifugation after 2, 4, and 6 h. The kinetics of cell-mediated plasminogen activation was determined by measuring amidolytic activity of the plasmin generated from plasminogen. The reaction was conducted with the substrate H-d-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide (Spectrozyme #251, American Diagnostica) at a final concentration of 100 μm. The reaction was initiated by the addition of substrate to 200 μl of conditioned media and was monitored at 405 nm in a PerkinElmer Life Sciences HTS 7000 Bioassay reader (Shelton, CT). Cell Invasion Assay—A cell invasion assay was conducted with QC-M™ 96-well cell invasion assay kit (Chemicon International). This well invasion plate is based on the Boyden chamber principle. This plate contains 96 inserts; each containing an 8-μm pore size polycarbonate membrane coated with a layer of ECMatrix™. Briefly, after detaching with cell dissociation solution (Sigma), CCL-222 cells were cultured in suspension in RPMI 1640 with 10% FBS for 2 h. Then, the cells were resuspended in serum-free RPMI 1640, and 5 × 104 cells were seeded into the extracellular matrix layer, which had been previously rehydrated at room temperature for 1–2 h. For the plasmin-dependent invasion assay, 0.2 μm plasminogen was added to the cell suspensions. 150 μl of RPMI 1640 media containing 10% fetal bovine serum was added to the lower chamber as chemoattractant. Cells were incubated for 24 h at 37 °C in a CO2 incubator (5% CO2). Invaded cells on the bottom of the insert membrane were dissociated from the membrane by incubation with cell detachment buffer and subsequently lysed and detected by CyQuant GR dye. The fluorescence was quantified with a fluorescence plate reader using a 485/535-nm filter set. Chamber Migration Assay—Migration was evaluated using a modified Boyden chamber assay. 8-μm cell culture inserts containing polyethylene tetrephthalate (BD Biosciences) were placed within a 24-well chamber containing 0.8 ml of RPMI 1640 medium with 10% FBS. 1.5 × 105 cells were seeded into the inserts suspended in 0.3 ml of serum-free RPMI 1640 media with 0.2 μm plasminogen. After incubation for 24 h at 37 °C in a CO2 incubator (5% CO2), the upper surface of the filter was scraped to remove non-migratory cells. Migrated cells were fixed with 4% PFA and stained with crystal violet. For quantification, the average number of migrating cells per field was assessed by counting 10 random fields under a light microscope (400×). Data indicate the mean obtained from three separate chambers. Miscellaneous Techniques—Protein concentrations were determined using Coomassie Brilliant Blue and BSA standards as described by Bradford (38.Bradford M.M. Analyt. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222437) Google Scholar). All reagents used were of analytical grade or better. Data were analyzed using Sigma Plot (Jandel Scientific). Colo 222 Colorectal Cells Express Extracellular S100A10 but Not Annexin A2—The CCL-222 cell line is a human intestinal tumor cell line that forms liver micrometastases in nude mice. To investigate the intracellular distribution of S100A10 and annexin A2, the colorectal cells were permeabilized and simultaneously stained for both S100A10 and annexin A2. As shown in Fig. 1A, immunofluorescence microscopic analysis established the presence of both S100A10 and its ligand annexin A2 within these cells. The distribution of S100A10 and annexin A2 immunofluorescence was consistent with the majority of these proteins colocalizing at the submembranous region of the cell. Confocal microscopy also confirmed that the majority of intracellular S100A10 and annexin A2 colocalized at the plasma membrane (Fig. 1C). Next, the immunofluorescence distribution of S100A10 and annexin A2 was examined in non-permeabilized colorectal cells (Fig. 1, B and D). We observed that S100A10 was present in discrete patches on the cell surface similar to structures observed for this protein on the extracellular surface of breast carcinoma, HT1080 fibrosarcoma, glioma, and human umbilical vein endothelial cells (32.Mai J. Finley Jr., R.L. Waisman D.M. Sloane B.F. J. Biol. Chem. 2000; 275: 12806-12812Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 34.Choi K.S. Fogg D.K. Yoon C.S. Waisman D.M. FASEB J. 2003; 17: 235-246Crossref PubMed Scopus (62) Google Scholar, 39.Kang H.M. Choi K.S. Kassam G. Fitzpatrick S.L. Kwon M. Waisman D.M. Trends. Cardiovasc. Med. 1999; 9: 92-102Crossref PubMed Scopus (53) Google Scholar). Surprisingly, we did not observe immunofluorescence staining for annexin A2, suggesting that annexin A2 was not present on the extracellular surface. Because the anti-annexin A2 antibody easily detected intracellular annexin A2, our inability to detect extracellular annexin A2 was not due to a problem with antibody reactivity. To confirm the absence of annexin A2 from the cell surface, cell surface proteins were biotinylated and isolated by avidin-Sepharose pull-down. The biotinylated protein fraction was analyzed by Western blotting. As shown in Fig. 1E, only S100A10 and not annexin A2 was detected in the biotinylated protein fraction. Thus, both immunofluorescence microscopy and surface biotinylation suggested that annexin A2 was not present on the surface of the colorectal cells. The absence of annexin A2 from the extracellular surface was unexpected, because typically both S100A10 and its binding partner, annexin A2, are found on the extracellular surface as the heterotetrameric complex, AIIt (26.Yeatman T.J. Updyke T.V. Kaetzel M.A. Dedman J.R. Nicolson G.L. Clin. Exp. Metastasis. 1993; 11: 37-44Crossref PubMed Scopus (94) Google Scholar, 31.Kassam G. Choi K.S. Ghuman J. Kang H.M. Fitzpatrick S.L. Zackson T. Zackson S. Toba M. Shinomiya A. Waisman D.M. J. Biol. Chem. 1998; 273: 4790-4799Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 32.Mai J. Finley Jr., R.L. Waisman D.M. Sloane B.F. J. Biol. 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Life Sci. 2001; 58: 902-920Crossref PubMed Scopus (749) Google Scholar), whereas uPA binds to its receptor, uPAR. As shown in Fig. 2A, S100A10 and uPAR share a similar distribution on the cell surface and appear to localize to one or two distinct regions of the cell surface. Confocal microscopy also confirmed the similar distribution of these proteins, although the distribution of S100A10 appeared to be more restricted than that of uPAR (Fig. 2B). At high magnification S100A10 and uPAR appear to colocalize in some regions of the cell surface (Fig. 2C). Interestingly, we also observed that the majority of plasminogen binding sites at the cell surface colocalized with uPAR (Fig. 2D). Collectively, these results suggest the presence of S100A10, uPAR, and plasminogen at a common locus on the extracellular surface. Silencing of the S100A10 Gene by RNA Interference—To study the role of S100A10 in plasminogen regulation we used the pSUPER system to stably suppress the expression of the S100A10 gene. The pSUPER construct consists of a H1-RNA promoter cloned next to the 19-nucleotide S100A10 sequence (nucleotides 199–217) separated by a short 9-nucleotide spacer that forms the hairpin, followed by the reverse compliment of the same nucleotide sequence. The pSUPER-Con vector was identical to the pSUPER-S100A10 vector except the 19-nucleotide sequence was derived from an irrelevant nucleotide sequence. CCL-22 colorectal cells were transfected with the pSUPER-S100A10 or pSUPER-Con, and cells were selected with puromycin and cultured under these conditions for about 1 month. As shown in Fig. 3,Fig. 3, both immunofluorescence microscopic analysis (Fig. 3, A–D,Fig. 3, A–D) and Western blotting (Fig. 3, E and F) showed that the S100A10 levels are reduced in the pSUPER-S100A10 cells compared with the pSUPER-Con cells. Furthermore, the annexin A2 levels were not affected by the S100A10 knockdown, establishing the specificity of the action of the S100A10 siRNA (Fig. 3F). In previous studies we used the pLIN vector to stably transfect HT1080 fibrosarcoma cells with a full-length antisense cDNA to S100A10 (34.Choi K.S. Fogg D.K. Yoon C.S. Waisman D.M. FASEB J. 2003; 17: 235-246Crossref PubMed Scopus (62) Google Scholar). It was interesting to note that this antisense strategy was unsuccessful in the CCL-222 cells. Although transfection of CCL-222 cells with the pLIN-S100A10 vector resulted in stable G418-resistant cells, as shown in Fig. 3E, the S100A10 protein levels were unchanged.Fig. 3S100A10 gene silen
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