1. Inactivating mutations of caspase-8 gene in colorectal carcinomas
Hong Sug Kim, Jong Woo Lee, Young Hwa Soung, Won Sang Park, Su Young Kim, Jong Heun Lee, Jik Young Park, Youg Gu Cho, Chang Jae Kim, Seong Whan Jeong, Suk Woo Nam, Sang Ho Kim, Jung Young Lee, Nam Jin Yoo, Sug Hyung Lee 
Gastroenterology 
Volume 125, Issue 3, Pages (September 2003)
DOI: /S (03)01059-X

2. Figure 1
Mutations of the caspase-8 gene in colon cancers. (A) SSCP and (B) sequencing analysis of caspase-8 DNA from tumors (lane T) and normal tissues (lane N). (A ) Arrows (lane T) indicate aberrant bands as compared with SSCP from normal tissue (N). (B) Arrows indicate nucleotide substitutions in tumor tissue as compared with normal tissue.
Gastroenterology  , DOI: ( /S (03)01059-X)

3. Figure 2
Defective apoptotic activities of tumor-derived caspase-8 mutants. (A) 293T cells were transfected with 1.3 μg of wild-type (WT) caspase-8 or each tumor-derived caspase-8 mutant together with 0.2 μg of pEGEF. Twenty-four hours after transfection, cells were fixed in 10% methanol for 15 minutes and stained with 4′,6-diamidino-2-phenylindole 1 μg/mL for 15 minutes, and the nuclei were examined by fluorescence microscopy (mean ± SD; n = 4). (B) 293T cells were co-transfected with Fas or TRAIL-R2 construct and a 4-fold excess of each mutant caspase-8. Cell death was analyzed as described in (B).
Gastroenterology  , DOI: ( /S (03)01059-X)

4. Figure 3
Impaired PARP cleavage by caspase-8 mutations. 293T cells were transfected with 1.3 μg of wild-type (WT) caspase-8 or each tumor-derived caspase-8 mutant. Ten hours after transfection, cell lysates were normalized for total protein content and then used by direct immunoblot analysis by anti-PARP antibodies (Pharmingen, San Diego, CA).
Gastroenterology  , DOI: ( /S (03)01059-X)

5. Figure 4
Decreased 5-fluorouracil (5-FU)-induced cell death by caspase-8 mutations. 5-FU (600 μmol/L) was added to DLD-1 cells 3 hours after transfection of the caspase-8 mutants; 24, 48, and 72 hours later, cell death was determined by 4′,6-diamidino-2-phenylindole staining.
Gastroenterology  , DOI: ( /S (03)01059-X)

6. Figure 5
Binding of caspase-8 mutants to FADD. 293T cells were cotransfected with expression constructs for human HA-FADD either with the Flag-caspase-8 dominant-negative or Flag-caspase-8 R413Q, Flag-caspase-8 R413 stop, or Flag-caspase-8 frameshift mutant as indicated. The transfected cells were harvested and lysed 1 day later, and immunoprecipitated with anti-Flag antibodies, and sequentially immunoblotted with anti-HA antibody as indicated. Aliquots of the same lysates (normalized for total protein content) were also analyzed directly by SDS-PAGE/immunoblotting as indicated. IP, immunoprecipitation; WB, Western blotting.
Gastroenterology  , DOI: ( /S (03)01059-X)

7. Figure 6
Visualization of caspase-8 expression in the tissues by immunohistochemistry. Antibodies were detected by a diaminobenzidine method that produces a brown color. Counterstaining of nuclei was performed with hematoxylin (blue). (A) Primary colon carcinoma cells show immunoreactivity for caspase-8 in the cytosol. Fibroblasts between the tumor nests are negative for the immunostaining. (B) Negative control of caspase-8 immunostaining (original magnification 200×).
Gastroenterology  , DOI: ( /S (03)01059-X)