Supplementary Material and Methods Quantitative RT-PCR (qrt-pcr) We performed RT-PCR, cloning, sequencing and qrt-pcr in murine melanoma cell lines and melanocytic tumors from RET-mice in accordance with the method previously described (10). The primer sequences for murine total Espin (detection of all Espin isoforms) were 5 -GCCTGGAGACGAGACAT-3 (forward) and 5 -AAGATGACCTGTCGCTG-3 (reverse) and those for glyceraldehydes-3-phosphate dehydrogenase (Gapdh) were 5 -CAAGAAGGTGAAGCA-3 (forward) and 5 -CCAGGAAATGAGCTTGCAC-3 (reverse). To clarify the expression of Espin isoforms, pairs of specific probes (Universal Probe Library: Roche) and primers were prepared. The Universal Probe Library system is based on TaqMan Probe technology. The expression levels of Espin isoforms and total Espin measured by real-time qrt-pcr were adjusted by the expression level of Gapdh. The probe and primer sequences were indicated probe number 83, 5 -TGCAGTGGCTGCTCACAC-3 (forward) and 5 -GGACTGTGGCACCAGAGTTATC-3 (reverse) for primer set 1, probe number 32, 5 -GAGGTGGACTCGCTCAAGG-3 (forward) and 5 -GCTTTGGAGTCCGCTGAG-3 (reverse) for primer set 2A-A, probe number 96, 5 -GGAGGTGGACTCGCTCAA-3 (forward) and 5 -CTCCGAATCCTGCCTGAG-3 (reverse) for primer set 2B-B, probe number 102, 5 -TCTAGGTGGGGGCCGTAT-3 (forward) and 5 -GTCGGCTTCAGGCTCTTG -3 (reverse) for primer set 4, and probe number 9, 5 -AGCTTGTCATCAACGGGAAG-3 (forward) and 5 -TTTGATGTTAGTGGGGTCTCG-3 (reverse) for primer set for Gapdh. Construction of plasmids. 1
Murine Espin-3A full-length cdna (GeneBank accession number: AY587570) was obtained by RT-PCR using KOD FX high fidelity DNA polymerase (TOYOBO) from RNAs extracted from the murine melanoma cell line B16F1 and murine testis organ. The Espin-3A cdna was inserted into Entry-Vector and converted to several expression vectors using the Gateway system (Invitrogen) according to the manufacturer s protocol. The p-dest17 (N-terminus 6xHis-Tag) expression vector was used for E. coli (DH5α and BL21) expression. All plasmids were sequenced. Immunoblotting. Western blotting was performed by the method previously described (Thang ND., PLoS One. 2011;6:e25636.). An anti-espin-ru rabbit polyclonal antibody was developed using peptides (SSSSTGSTKSFN) in this study (Supplementary Fig. S1). The anti-espin-ru rabbit polyclonal antibody recognized the sequences between exon r and u except for exon s and t products in Espin (Supplementary Figure S1), which are common in humans and mice. The sequences of exon s were original sequences in Espin isoforms 1, 2A and 2B. The peptides of exon t were original sequences in Espin isoform 4. Therefore, this antibody can detect only Espin isoforms 2A 2B 3A and 3B. The antibody IgG species were purified by using a Melon Gel IgG purification Kit (PIERCE) according to the manufacturer s protocol. Detection of Espin-3A protein by the antibody was confirmed (Supplemental Figure 3). Rabbit polyclonal antibodies against phosphorylated tyrosine 397 in FAK (Invitrogen) and phosphorylated tyrosine 181 in Paxillin (EPITOMICS) and mouse monoclonal antibodies against Gapdh (Cell Signaling Technology, Inc.), FAK (BD Transducton Laboratories), Vinculin (SIGMA) and Paxillin (BD) were used as primary antibodies. For detection of 6xHis fusion protein, a Universal His Western blot Kit (Clontech) was used according to the manufacturer s protocol. Densitometric evaluation 2
was performed using the software program WinROOF (MITANI Corporation) according to the method previously described (Yajima I et al., Arch Toxicol. 2012;86:961-73.). Establishment of shrna-stable clones. Stable Espin knockdown clones were established by the method previously described (Song JH et al., Cancer Res. 2007;67:6946-55) with some modifications. Briefly, Mel-ret cells were transfected with prnat-u6.1/neo sirna expression plasmid (GeneScript Corporation) containing a GFP expression gene with either sirna targeting Espin or a scrambled control sequence. The targeted sirna sequences were 5 -CAACGGGUGAUAACUCAGAGCUUCU-3 for the murine Espin sirna sequence and 5 - CAAGUGGAAUAACUCCGAGUGCUCU-3 for the control sirna sequence (only sense sequences shown). GFP-positive cells were collected by a cell sorter (Becton-Dickison) after selection of stable clones. Rho kinase activity determined by a pull-down assay Pull down assays were performed by using RhoA and Rac1 Activation Assay Kits (CELL BIOLABS) according to the manufacturer s protocol. shrna stably expressed Mel-ret cells were seeded on type I collagen (10 µg/ml)-coated 10 cm culture plates. The subconfluent cells were lysed, and then GTP-bound active RhoA was pulled down by Rhotekin Rho-binding domain agarose beads and GTP-bound active Rac1 was pulled down by PAK1 p21-binding domain agarose beads. Cell lysates in a buffer (25mM HEPES ph 7.5, 150 mm NaCl, 1% Nonidet P-40, 10 mm MgCl2, 1 mm EDTA, 2% glycerol) plus protease inhibitors and the pull-down fraction were separated on 12% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and then immunoblotted with anti-rhoa and anti-rac1 mouse monoclonal antibodies. 3
Immunofluoresense Mel-ret cells were plated on type I collagen (10 µg/ml)-coated glass dishes (Nunc). Cells were fixed with 4% paraformaldehyde (PFA) in posphate buffered saline (PBS) and treated with 0.1% Triton X-100/PBS followed by blocking with 1% bovine serum albumin/pbs. Cells were labeled with anti-espin-ru polyclonal antibody diluted 1: 100 and then labeled with Alexa-488 conjugated anti-rabbit IgG secondary antibody diluted 1: 1000 (Invitrogen). Cell cytoskeleton F-actin was labeled with Alexa-568-conjugated phalloidin. Fluorescence was examined using a confocal laser-scanning microscope (Fluoview FV1000 software, Olympus MF1000). Alexa568 signal (red) was converted to magenta color by Adobe Photoshop software. Immunohistochemical staining Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections (3 µm in thickness) of human melanocytic nevi and melanoma. Human tissue array slides were deparaffinized in xylene and dehydrated through a graded series of alcohols and then boiled at 95 degrees in 0.01 M citrate acid buffer (ph6.0) for 20 minutes. For de-melanization, slides were treated with 0.25% KMnO4 in H2O for 60 minutes and then treated with 5% oxalic acid for 5 minutes. The slides were incubated in 3% H2O2 in methanol for 15 minutes and were blocked by 1.5% normal goat serum in PBS for 20 minutes. Anti-Espin-ru polyclonal antibody was diluted at 1:50 for 60-minute incubation. Biotinylated anti-rabbit IgG secondary antibody was reacted at a dilution of 1:150 for 30 minutes. Then the slides were stained using the avidin-biotin complex immunoperoxidase method (Vector VIP substrate kit: Vector). Counterstaining was performed using methyl green (Vector). Slides were rinsed with PBS after each stage of the 4
procedure. Visible tumors (GFP-positive) and the lungs were excised for histological examination on our xenograft study. Harvested tissues were fixed in 4% PFA, embedded in paraffin and sectioned at 5 μm, and then HE staining was performed. Statistical analysis Statistical analysis in this study was performed according to the method previously described (Kato M et al., Cancer Epidemiol Biomarkers Prev 2011;20:1622-8.). Results from more than three independent experiments in each group were statistically analyzed by Student's t-test. In the case of ESPIN immunohistochemistry in human melanocytic lesions, statistical significance of ratio was determined by Fisher s exact test. We used the SPSS (version 18) software package (SPSS Japan Inc.) for statistical analyses, and the significance level was set at p< 0.05. 5