Toso is a negative regulator of CD95-induced apoptosis. (A) The modulation of Toso surface expression by Toso knock-down or overexpression. Representative flow cytometric analysis of BJAB cells stably expressing Toso specific miRNA (blue), control miRNA (black) or hToso cDNA (red). (B) Toso knock-down results in an increased sensitivity to CD95L-induced apoptosis. BJAB cells stably expressing control miRNA (black) or 2 different Toso specific miRNAs (red) were stimulated with various concentrations of CD95L for 5 hours. Apoptotic cells were assessed by staining with annexin V and relative apoptosis rates (%) were quantified. Representative data from > 10 independent experiments are shown. (C) Toso overexpression protects from CD95L-induced apoptosis. BJAB cells expressing either a control vector (black) or hToso cDNA (red) were stimulated for 5 hours with different concentrations of CD95L. Relative apoptosis was analyzed as in (B). Data shown are representative of more than 10 experiments. (D) CD95L-induced apoptosis in Toso knock-down primary T cells. Activated human peripheral blood T cells were lentivirally transduced with Toso specific miRNA/eGFP (black bars) or control miRNA/eGFP (white bars). Cells were stimulated with CD95L and apoptosis was assessed by staining with annexinV. Apoptotic cells were analyzed in GFP⁺ (transfected) and GFP⁻ (nontransfected) cell populations. Data shown are representative of 3 experiments. Statistical analysis was performed by 2-tailed Student t test (*P < .05, **P < .01). (E) CD95L-induced apoptosis in Toso overexpressing primary T cells. Activated human peripheral blood T cells expressing a Toso-IRES-GFP construct (black bars) or control DNA (white bars). Apoptotic cells were analyzed by staining with annexinV in GFP+ (transfected) and GFP- (nontransfected) populations. Data shown are representative of 3 experiments. (F) Reconstitution of CD95-induced apoptosis in Toso knock-down cells. BJAB cells stably expressing Toso specific miRNAs were transiently transfected with a ‘miRNA resistant’ Toso cDNA containing silent mutations in the specific miRNA target site. Apoptosis was induced by CD95L stimulation for 5 hours. Flow cytometric histograms of mock treated control cells (gray filled) or Toso silent mutant expressing cells (red) are shown on the left. Relative percent of apoptosis is quantified on the right. Data shown are representative of 2 experiments.