Figure 5

Functional analyses of Medaka Fas, FADD and Casp8. (A) Schematic diagram of the plasmid constructs for the expression of human and Medaka chimeric Fas (h/oFas), Flag/oFADD-EGFP and EGFP/oCasp8 proteins. The chimeric h/oFas consists of the extracellular domain of human FAS and the transmembrane and cytoplasmic regions of Medaka Fas. The Flag/oFADD-EGFP construct translates both Flag-tagged Medaka FADD and EGFP molecules from a bicistronic mRNA. The EGFP/oCasp8 is a fusion of Medaka caspase-8 with EGFP at the N-terminus. (B) Cytotoxicity assays of chimeric Fas introduced into mouse NIH3T3 cells. Empty pME18S vector (panels a and b), pME18S-h/oFas (panels c, d and e) or pME18S-hFAS (panels f and g) was cotransfected transiently with pEGFP-C1 into NIH3T3 cells. After culture for 48 h, these transfectants were incubated for 14 h in the presence (panels b, d, e and g) or absence (panels a, c and f) of 500 ng/ml anti-human Fas antibody CH11. Cell viability was measured by detecting EGFP-positive cells by fluorescent microscopy. Arrows indicate dead cells. The typical dead cell exhibiting apoptotic bodies was magnified (panel e). (C) Immunocytochemical analysis of transfectants expressing h/oFas. pME18S-h/oFas (panels a and b) or pME18S-hFAS (panels c and d) were cotransfected transiently with phLBR1TM-EGFP into NIH3T3 cells. After culturing for 48 h, transfectants were incubated for 12 h in the presence (panels b and d) or absence (panels a and c) of CH11. Activated Casp3 in cells expressing EGFP in the nucleus was visualized by staining with anti-cleaved Casp3 and fluorescently-labeled secondary antibodies. After counterstaining with DAPI, cells were photographed by fluorescent microscopy. Arrows indicate transfectants. (D) Cytotoxicity assays of Medaka FADD-expressing mammalian cell lines. The pME18S-Flag/oFADD-EGFP plasmid was transfected into HeLa cells (panels a and b) and wild-type (panel c) or Casp8-deficent (panel d) MEF cells. Half of the HeLa transfectants were cultured in the presence of 100 μM zVAD-fmk (panel b). After 24 h of culture, cells were washed, fixed, and examined by fluorescence microscopy. Viable cells were defined as EGFP-positive cells, while typical dead cells are shown by arrows. Abbreviations: WT, wild-type; Casp8-KO, Casp8-deficient. (E) Cytotoxicity assays of Medaka Casp8-expressing HeLa cells. The pCMV-EGFP/oCasp8 construct, encoding EGFP/oCasp8, was transfected into HeLa cells alone (panels a, b and d) or in conjunction with pCX-CrmA that encoded CrmA (panel c). Half of transfectants expressing EGFP/oCasp8 alone were incubated with 100 μM zVAD-fmk (panels b and d). After 24 h of culture, transfectants were washed, fixed, and examined by fluorescence microscopy. Viable cells were defined as EGFP-positive cells. Surviving cells expressing EGFP/oCaspa8 were examined by confocal laser scanning microscopy (panel d). In panel d, a dotted line demarks the edge of a single cell. (F) The DNA content of transfectants expressing Medaka FADD or caspase-8 was assessed by flow cytometry. Twenty-four hours after transfection, the DNA content of cells transfected with pME18S (panel a), pME18S-Flag/oFADD-EGFP (panels b and c), pCMV-EGFP/oCasp8 (panels d and e) together with pCX-p35 (panels c and e) was analyzed by staining with PI. The percentage indicates the cellular population with sub-G₁ DNA content.