Supplementary MaterialsS1 Fig: expression in melanoma cell lines. significantly reduced correlating with the decrease of cellular Ust protein. Single cell migration of B16VshUst(16) cells showed a decreased cell movement phenotype. The adhesion of B16V cells to fibronectin depended on 51 but not v3 integrin. Inhibition of glycosaminoglycan sulfation or blocking fibroblast growth factor receptor (FgfR) reduced 5 integrin in B16V cell lines. Interestingly, FgfR1 expression and activation was reduced in knock-down cells. knock-down in human melanoma cells also showed a reduction in is involved in metastasis of breast cancer [15] and JW 55 the chondroitin 4,6-O sulfotransferase encoded by in Lewis lung carcinoma (LCC) [16, 17]. However, Ust (small letters, because mouse) has not been studied in this context. Interestingly, B16 melanoma cells have 1.5 times more DS compared to LCC [18] suggesting that 2-O sulfation of CS/DS might play an important role in melanoma metastasis. Previous reports showed that CS/DS affects cell adhesion and migration [7, 19] and that the lack of l-IdoUA on the cell surface leads to an impaired directed cell migration [20]. In the central nervous system, JW 55 a tissue rich in CS-proteoglycans, over-sulfated CS are involved in neuronal migration and axon regeneration [19, 21]. Recently, a reduction in has been reported for siRNA-mediated versican knock-down in a leiomyosarcoma smooth muscle cell line [22]. Furthermore, the lack of Ust in skin of decorin-deficient mice impairs Fgf2 and Fgf7 binding and keratinocyte differentiation [23]. The occurrence of 2-O sulfated cell surface CS/DS can tune the Fgf2-mediated effect on cell migration of CHO cells and fibroblasts [5, 23]. A critical strep in migration is cell adhesion which is JW 55 mainly mediated via integrins, heterodimeric cell surface receptors which mediate bidirectional signaling between cells and the extracellular matrix (ECM). During cell migration the JW 55 function of 51 integrin and v3 integrin is tightly Rabbit Polyclonal to STA13 regulated [24]. The role of 5 integrin in cancer progression is controversial [25]. 5 integrin also plays an important role in melanoma cell motility since its upregulation enhances migration [26, 27]. This is further supported by findings that human carcinomas frequently express high levels of 51 integrin which had been correlated with a more aggressive carcinoma phenotype [25]. For B16F10 melanoma cells a direct correlation of the metastatic potential and increased 5 integrin function was demonstrated [28]. The aim of the present study was to demonstrate that Ust is a critical regulator of melanoma cell adhesion and motility and data showed that B16VshUst(16) cells have a significantly reduced pulmonary metastatic potential. Therefore, we can link for the first time Ust and CS/DS 2-O sulfation with 5 integrin expression, an important factor for metastasis of melanoma cells. Materials and Methods Materials The following primary antibodies were used: UST D-20 (Santa Cruz Biotechnology), -actin, anti 5 integrin, anti 1 integrin (Millipore), Alexa Fluor? 647 anti-mouse CD49e, LEAF? 1, 5, v and 3 integrin blocking antibodies (anti-mouse, BioLegend, California, USA) anti-rabbit-HRP secondary antibody (GE Healthcare, UK). F-actin was visualized by Alexa488-conjugated phalloidin (Invitrogen, USA). PD173074, fibronectin, mouse-Fgf2, chondroitin 6-sulfate (CS-6S) (Sigma Aldrich, Deisenhofen, Germany), chondroitin ABC lyase and heparitinase mix (heparinase II/III, 4:1) (Amsbio, UK). Cell culture Murine melanoma (B16V) cells [29] were grown to confluence in bicarbonate buffered RPMI 1640 (Sigma) supplemented with 10% (v/v) bovine serum (FBS) at 37C in a humidified atmosphere of 5% CO2. Of note, B16V cells display a black color due to their melanin. All experiments were performed at passages where cells contained melanin. Human HT168-M1, HT199 [30] and MV3 [31] melanoma cells were grown in RPMI 1640 with 10% (v/v) FBS and cultured as described before. Knock-down of in melanoma cells B16V cells were stably transfected with shRNA-Ust(m) plasmid as a pool of 3 target-specific lentiviral vector plasmids, each encoding 19C25 nt (plus hairpin) shRNAs designed to knock-down gene expression (Santa Cruz), following the manufacturers protocol. Control cells were mock transfected with shRNA.