Fig. (4) SG-12 promoted the progression of early stage of Fasmediated cell death. (A) A20/2J cells (1 × 10⁶ cells/ml) were incubated without or with 100 ng/ml anti-mouse Fas mAb, 5 µg/ml F(ab’)2 of anti-mouse IgG pAb, or 50 µM z-VAD-fmk in the presence or absence of SG-12 for 4 h. Fractions of apoptotic cells (Annexin V⁺PI^-) are shown. Values are means ± SD from triplicate samples. Statistically significant differences (**p< 0.01) with respect to the values without SG-12 are indicated. (B) A20/2J cells (1 × 10⁶ cells/ml) were incubated without or with anti-mouse Fas mAb and 5 µg/ml F(ab’)₂ of anti-mouse IgG pAb in the presence or absence of 8 µM SG-12. After 4 h, cells were lysed and proteins were detected by immunoblotting as described in “Materials and Methods.” (C) A20/2J cells (1 × 10⁸ cells/ml) were preincubated without or with 8 µM SG-12 or 4 µM LY294002 for 5 min and stimulated with an F(ab’)₂ of an anti-mouse IgG pAb (2.5, 5, 10 µg/ml) for 5 min. Then, the cells were lysed and proteins were detected by immunoblotting as described in “Materials and Methods.” Essentially the same result was obtained in another independent experiment.