Series using a certain combination of the exon specific primers, which covered the whole variable region with five overlapping sequences. The first primer pair was located on the two standard regions allowing detection of all variable exons (S5′?S3′, primer pair 1). The second was on exon 4 of the standard region and v3, which makes possible to detect v2-v3 co-expression (S5′-v3′, primer pair 2). The third straddles the v3-v10 region by binding to v3 and exon 16 of the standard region (v35′-S3′, primer pair 3). The fourth pair was designed to exon 4 and v6 detecting the co-expression of v2 5 with v6 (S5′-v63′, primer pair 4). The fifth one was detecting v4 5 expression of v3 6 co-expressing isoforms by binding to v3 
and v6 (v35′-v63′, primer pair 5). The five PCR products of the same sample were run always in this same order in every case, so the pattern of the bands were comparable across all of our samples in the different experimental models. This bar code-like pattern is what we define as the `fingerprint’, a simplified representation of the CD44 ASP. With this method we examined the CD44 ASP of human melanoma cell lines (HT168M1, WM35, WM983B, A2058 and HT199) in MedChemExpress 10236-47-2 culture to establish whether there is a pattern that is conserved across these genetically different tumours. As it is shown in Fig. 3A and 4, we found 
a consistent pattern throughout, which we refer to as the melanoma fingerprint. Some of the isoforms,Quantitative PCR AnalysisFor quantitative measurement of the expressed CD44 variable exons q-PCR reactions were used (iQ SYBRH Green Supermix,Bio-Rad), by cycling conditions 3 min at 95uC, then 40 cycles at 95uC 30 sec, 55uC 30 sec, 72uC 1 min. Starting quantities were defined on the basis of standard fivefold dilution series (1x-625X)CD44 Alternative Splicing Pattern of MelanomaFigure 1. CD44 isoforms validated by next generation sequencing. A. CD44 isoforms from the qualitative picture of pairing the variable exon specific primers with the standard region specific ones both 59 and 39 directions in HT168 human melanoma cell line. These isoforms were validated by next generation sequencing. B. Further validated isoforms from next generation sequencing with the primer pairs of the fingerprint. doi:10.1371/journal.pone.0053883.gwhich were predicted from the melanoma fingerprint, based on the size of the bands, were confirmed by next generation sequencing. Although the reading frame of 454 GS Junior is significantly wider than that of similar 15755315 techniques, its ITI-007 higher limit is still 400?00 bp. Therefore, even though next generation sequencing is rather promising, we are still relying on estimations in the case of larger products. 10 isoforms were confirmed (Fig. 1A) and a further 26 predicted (Figure S5) as part of the melanoma CD44 fingerprint. We then compared this pattern to that of other human tumour cell lines grown in culture. These included cell lines derived from human colorectal adenocarcinoma (HT29, HT25, HCT116), human oral squamous cell carcinoma (PE/CA PJ15 and PE/CA PJ41), vulval squamous cell carcinoma (A431) and K562 human erythromyeloblastoid leukemia cell lines.Comparison was also made with primary cultured human melanocytes, skin keratinocytes and skin fibroblasts (Fig. 4). In each case the fingerprint differed unambiguously from the melanoma fingerprint, raising the possibility of a melanoma specific isoform expression pattern.xenograft variant of A2058; HT168M1, a cell line which is the in vivo selected metast.Series using a certain combination of the exon specific primers, which covered the whole variable region with five overlapping sequences. The first primer pair was located on the two standard regions allowing detection of all variable exons (S5′?S3′, primer pair 1). The second was on exon 4 of the standard region and v3, which makes possible to detect v2-v3 co-expression (S5′-v3′, primer pair 2). The third straddles the v3-v10 region by binding to v3 and exon 16 of the standard region (v35′-S3′, primer pair 3). The fourth pair was designed to exon 4 and v6 detecting the co-expression of v2 5 with v6 (S5′-v63′, primer pair 4). The fifth one was detecting v4 5 expression of v3 6 co-expressing isoforms by binding to v3 and v6 (v35′-v63′, primer pair 5). The five PCR products of the same sample were run always in this same order in every case, so the pattern of the bands were comparable across all of our samples in the different experimental models. This bar code-like pattern is what we define as the `fingerprint’, a simplified representation of the CD44 ASP. With this method we examined the CD44 ASP of human melanoma cell lines (HT168M1, WM35, WM983B, A2058 and HT199) in culture to establish whether there is a pattern that is conserved across these genetically different tumours. As it is shown in Fig. 3A and 4, we found a consistent pattern throughout, which we refer to as the melanoma fingerprint. Some of the isoforms,Quantitative PCR AnalysisFor quantitative measurement of the expressed CD44 variable exons q-PCR reactions were used (iQ SYBRH Green Supermix,Bio-Rad), by cycling conditions 3 min at 95uC, then 40 cycles at 95uC 30 sec, 55uC 30 sec, 72uC 1 min. Starting quantities were defined on the basis of standard fivefold dilution series (1x-625X)CD44 Alternative Splicing Pattern of MelanomaFigure 1. CD44 isoforms validated by next generation sequencing. A. CD44 isoforms from the qualitative picture of pairing the variable exon specific primers with the standard region specific ones both 59 and 39 directions in HT168 human melanoma cell line. These isoforms were validated by next generation sequencing. B. Further validated isoforms from next generation sequencing with the primer pairs of the fingerprint. doi:10.1371/journal.pone.0053883.gwhich were predicted from the melanoma fingerprint, based on the size of the bands, were confirmed by next generation sequencing. Although the reading frame of 454 GS Junior is significantly wider than that of similar 15755315 techniques, its higher limit is still 400?00 bp. Therefore, even though next generation sequencing is rather promising, we are still relying on estimations in the case of larger products. 10 isoforms were confirmed (Fig. 1A) and a further 26 predicted (Figure S5) as part of the melanoma CD44 fingerprint. We then compared this pattern to that of other human tumour cell lines grown in culture. These included cell lines derived from human colorectal adenocarcinoma (HT29, HT25, HCT116), human oral squamous cell carcinoma (PE/CA PJ15 and PE/CA PJ41), vulval squamous cell carcinoma (A431) and K562 human erythromyeloblastoid leukemia cell lines.Comparison was also made with primary cultured human melanocytes, skin keratinocytes and skin fibroblasts (Fig. 4). In each case the fingerprint differed unambiguously from the melanoma fingerprint, raising the possibility of a melanoma specific isoform expression pattern.xenograft variant of A2058; HT168M1, a cell line which is the in vivo selected metast.
