Forms at mRNA LevelWe visualized the expression of CD44 variable exons

Forms at mRNA LevelWe visualized the expression of CD44 variable exons in HT168 human melanoma by performing PCR reactions pairing the sense (59) primers of variable exons with the common antisense (39) primer localized on exon 16 and variable exon’s antisense (39) primers with the common sense (59) on the standard exon 4. Our results showed, that all the variable exons, which are considered variable in databases (v2-v10) were present. Also, this method with the overlapping sequences allowed us to construct some of the isoforms (Fig. 1 and Fig. S5), although, this still seems rather inaccurate as some of the exons seemed to have been of slightly different size. This size difference can possibly be explained by the fact that by next generation sequencing on the same tumour, we identified a daunting number of small deletions across the CD44 isoforms (data not shown). We made further attempts and cloned our PCR products from A2058 and HT168 M1 human melanoma cell lines, which resulted in certain isoforms being more dominant and inserting at a higher rate, but yet again, the full set of the expected/calculated isoforms could not be identified. However, direct sequencing of some of the cloned sequences confirmed that v1, is in fact missing in some of the isoforms, which tied in nicely, with our above mentioned PCR-based results (Fig. 2A). Furthermore, some isoforms contained a truncated version of v1 (Fig. 2B).Culturing on Different Gracillin MatricesFibronectin, laminin, collagen IV Matrigel, hyaluronate (each 50 mg/ml) and 0,9 NaCl solution (as control) were administered into different wells of a 6-well plate. After 3 hours of incubation on RT, supernatants were removed. 1? ml of 56104 cell/ml suspensions of HT168M1 was administered on the prepared matrix-films. After 72 hours of incubation, we removed supernatants, washed cell-films with EDTA, up-digested cell-films with tripsin-EDTA, collected up-grown cell suspensions and extracted total-RNA of cell masses with TRI-Reagent method.Metastasis Models Using scid MiceThis study was carried out in strict accordance with the recommendations and was approved by the Semmelweis University Regional and Institutional Committee of Science and Research Ethics (TUKEB permit number: 83/2009). All surgery was performed under Nembutal anaesthesia, and all efforts were made to minimize suffering. Cultured HT199 and HT168M1 human tumour cells were injected subcutaneously (5×105/50ml Gracillin web volume) at the same lower back 1662274 localisation into 10 newborn and 10 adult scid mice as well as intravenously into 5 adult scid mice for both cell line. On the 30th day, the animals were sacrificed by bleeding under anaesthesia. Primary in vitro cell cultures were formed from the primary tumour, circulating tumour cells and the lung metastases of the same animal implanted as a newborn. Also, the primary tumour, circulating tumour cells and the i.v. transplanted lung colonies from the adult animals were used to create cell cultures the same way (Figure S4). For comparative measurements the different tumours, i.e. primary tumour, circulating tumour cells, lung metastasis, always derived from the same animal to allow standardisation of the host.The CD44 Melanoma FingerprintIn light of the complexity of CD44 isoform expression simple method to represent this pattern was developed which included v3 and v6?the exons considered to be of importance for melanoma progression. For this purpose, we designed a five primer pair containing PCR-reaction.Forms at mRNA LevelWe visualized the expression of CD44 variable exons in HT168 human melanoma by performing PCR reactions pairing the sense (59) primers of variable exons with the common antisense (39) primer localized on exon 16 and variable exon’s antisense (39) primers with the common sense (59) on the standard exon 4. Our results showed, that all the variable exons, which are considered variable in databases (v2-v10) were present. Also, this method with the overlapping sequences allowed us to construct some of the isoforms (Fig. 1 and Fig. S5), although, this still seems rather inaccurate as some of the exons seemed to have been of slightly different size. This size difference can possibly be explained by the fact that by next generation sequencing on the same tumour, we identified a daunting number of small deletions across the CD44 isoforms (data not shown). We made further attempts and cloned our PCR products from A2058 and HT168 M1 human melanoma cell lines, which resulted in certain isoforms being more dominant and inserting at a higher rate, but yet again, the full set of the expected/calculated isoforms could not be identified. However, direct sequencing of some of the cloned sequences confirmed that v1, is in fact missing in some of the isoforms, which tied in nicely, with our above mentioned PCR-based results (Fig. 2A). Furthermore, some isoforms contained a truncated version of v1 (Fig. 2B).Culturing on Different MatricesFibronectin, laminin, collagen IV Matrigel, hyaluronate (each 50 mg/ml) and 0,9 NaCl solution (as control) were administered into different wells of a 6-well plate. After 3 hours of incubation on RT, supernatants were removed. 1? ml of 56104 cell/ml suspensions of HT168M1 was administered on the prepared matrix-films. After 72 hours of incubation, we removed supernatants, washed cell-films with EDTA, up-digested cell-films with tripsin-EDTA, collected up-grown cell suspensions and extracted total-RNA of cell masses with TRI-Reagent method.Metastasis Models Using scid MiceThis study was carried out in strict accordance with the recommendations and was approved by the Semmelweis University Regional and Institutional Committee of Science and Research Ethics (TUKEB permit number: 83/2009). All surgery was performed under Nembutal anaesthesia, and all efforts were made to minimize suffering. Cultured HT199 and HT168M1 human tumour cells were injected subcutaneously (5×105/50ml volume) at the same lower back 1662274 localisation into 10 newborn and 10 adult scid mice as well as intravenously into 5 adult scid mice for both cell line. On the 30th day, the animals were sacrificed by bleeding under anaesthesia. Primary in vitro cell cultures were formed from the primary tumour, circulating tumour cells and the lung metastases of the same animal implanted as a newborn. Also, the primary tumour, circulating tumour cells and the i.v. transplanted lung colonies from the adult animals were used to create cell cultures the same way (Figure S4). For comparative measurements the different tumours, i.e. primary tumour, circulating tumour cells, lung metastasis, always derived from the same animal to allow standardisation of the host.The CD44 Melanoma FingerprintIn light of the complexity of CD44 isoform expression simple method to represent this pattern was developed which included v3 and v6?the exons considered to be of importance for melanoma progression. For this purpose, we designed a five primer pair containing PCR-reaction.

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