Volume 125, Issue 5, Pages (November 2003)

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Volume 125, Issue 5, Pages 1563-1564 (November 2003) A loss of function mutation in WISP3 derived from microsatellite unstable gastric carcinoma Shinji Tanaka, Keishi Sugimachi, Shin-Ichiro Maehara, Mitsuo Shimada, Yoshihiko Maehara Gastroenterology Volume 125, Issue 5, Pages 1563-1564 (November 2003) DOI: 10.1016/j.gastro.2003.04.013

Figure 1 (A) Schematic structures of WISP3(A)9 and WISP3(A)8. WISP3 protein is composed of the signal sequences (SS) (a black box), domain I homologue to IGF binding proteins (IGF-BP), domain II homologue to Von Willebrand factor type C repeats (VWC), domain III homologue to the thrombospondin type I domain (THBS), and domain IV of the cysteine knot domain (CK) (large boxes). Conserved cysteine residues are indicated by thin bars. The WISP3(A)8, derived from microsatellite unstable gastric carcinomas, lacks both the THBS and CK domain. The WISP3(A)8 sequences were deposited with GenBank, accession number AB075040. (B) Ectopic expression of WISP3 in Kato-III human gastric carcinoma cells. Stable transfectants of WISP3(A)9 cDNA or WISP3(A)8 cDNA were prepared with a bicistonic expression vector pIRESneo (Clontech, Palo Alto, CA), that contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus. The expression cassette contains a single promoter, which, in combination with the IRES, permits the translation of both the gene of interest and the neomycin-resistant gene from the same mRNA. Specific expression of WISP3 was detected in cell lysates of the transfectants using a polyclonal antibody against the WISP3 IGF-BP domain (sc-8871; Santa Cruz Biochemistry, Santa Cruz, CA). The mock-vector transfectants as a negative control. The molecular weights shown in the left lane. (C) Matrigel invasion assays on the transfectants or the parental gastric carcinoma Kato-III cells. The number of cells invading into the Matrigel matrix were determined for each clone, following 24-hour stimulation of chemotaxis by serum. To avoid clonal deviation, 3 independent clones of WISP3(A)9 or WISP3(A)8 transfectants were used, and the results were derived from 3 individual experiments. Error bars indicate the standard deviation from the mean. Gastroenterology 2003 125, 1563-1564DOI: (10.1016/j.gastro.2003.04.013)