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Supplementary Information Wnt5a signaling is involved in the aggressiveness of prostate cancer and expression of metalloproteinase Hideki Yamamoto, Naohide Oue, Akira Sato, Yasuhisa Hasegawa, Hideki Yamamoto, Akio Matsubara, Wataru Yasui, and Akira Kikuchi Supplementary material and methods Material and chemicals Anti-phospho- PKD/PKC (Ser742), anti-Rac1, anti-MMP-1, and anti-phospho-SAPK/JNK (Thr183/Tyr185) antibodies were obtained from Upstate (Temecula, MA), BD Transduction Laboratories (Bedford, MA), Daiichi Fine Chemical (Toyama, Japan), and Cell Signaling Technology (Beverly, MA), respectively. Anti-JunD (sc-74) and anti-JNK1 ((C-17)-G) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Other materials were obtained from commercial sources. Small interfering RNA (siRNA) In analyses with siRNA for Wnt4, Wnt5a, Wnt5b, Wnt7a, Frizzled (Fz) 2, Fz6, Fz7, Ror2, MMP-1, and Rac1, the following target sequences were used. Wnt4, 5’-GGTCAGGATGCTCTGACAA-3’and 5’-GCTCTGACAACATCGCCTA-3’; Wnt5a, 5’-CTGTGGATAACACCTCTGTTT-3’ and 5’-CTGTTCAGATGTCAGAAGTAT; Wnt5b, 5’-GAGGAAGCTGTGCCAATTGTA-3’ and 5’-GTGTATAAGATGGCAGACGTA-3’; Wnt7a, 5’-TGGGCGCAAGCATCATCTGTA-3’ and 5’-CGGCTTCGCCAAGGTCTTTGT-3’ ; Fz2, 5’-CGGTCTACATGATCAAATA-3’; Fz6, 1 5’-GGTTCCACCTTGTCGTAAA-3’; Fz7, 5’-TCACCTACCTGGTGGACAT-3’; Ror2, 5’-GCAACCTTTCCAACTACAA-3’ ; MMP-1, 5’- GCGTGTGACAGTAAGCTAA-3’; Rac1, 5’-GTGATTTCATAGCGAGTTT-3’, 5’-GTAGTTCTCAGATGCGTAA-3’, 5’-ATGAAAGTGTCACGGGTAA-3’, and 5’-GAACTGCTATTTCCTCTAA-3’; scrambled siRNA, 5’-CAGTCGCGTTTGCGACTGG-3’. The duplexes of RNA oligos were transfected into the cells using Oligofectamine (Invitrogen, Carlsbad, MA). Reverse transcription-PCR (RT-PCR) Semi-quantitative RT-PCR was performed as described previously (Hino et al., 2005). Forward and reverse primers were shown in Supplementary Table S3. Supplementary Reference Hino S, Tanji C, Nakayama K-I, Kikuchi A (2005). Phosphorylation of -catenin by cyclic AMP-dependent protein kinase stabilizes -catenin through inhibition of its ubiquitination. Mol. Cell. Biol. 25: 9063-9072. 2 Supplementary Figure legends Figure S1. siRNA for Wnt5a ,Wnt7a, and Wnt4 in PCa Cells. The levels of Wnt5a and Wnt7a mRNA in LNCap (a) and DU145 (b) cells transiently transfected with scrambled, Wnt5a, Wnt7a, or Wnt4 siRNA were quantified. Relative mRNA levels were expressed as the percentages of mRNA levels in cells without transfection of siRNA or transfected with scrambled siRNA. Figure S2. The expression profile of Frizzled (Fz) family members in PCa cells. (a) The mRNA levels of various Fzs in PC3, DU145, and LNCap cells were quantified by quantitative RT-PCR. The primers used for quantitative RT-PCR are described in the supplementary Table S3. (b) The mRNA levels of Wnt receptors in DU145 cells transiently transfected with scrambled, Fz2, Fz6, Fz7, or Ror2 siRNA were quantified. Relative mRNA levels were expressed as percentages of the mRNA levels in cells transfected with scrambled siRNA. 3