Download Purified Wnt-5a exerts antagonistic actions for Wnt-3a

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,
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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.
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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.
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