Figure 5From: The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fishFunctional 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.Back to article page