Figure 4

CTLA4-FasL effect on pro and anti apoptotic signals. (A) Sk-Hep1 (left) and A498 (right) cell lines were pre-incubated with or without caspase inhibitors (Z-VAD-FMK (general), Z-LEHD-FMK (caspase 9), Z-IETD-FMK (caspase 8)) for 1 hour followed by incubation with his₆CTLA4-FasL at different concentrations for 24 hours. Cells' viability was tested by the MTS assay. The results represent the average of three independent experiments. +/- SE (*p≤0.05). (B) Sk-Hep1 cells were incubated with CTLA4-FasL, sFasL, CTLA4-Ig or combination of the later two for 24 hours. Cell viability was tested by the MTS assay. The results represent the average of four independent experiments. +/- SE (*p ≤ 0.05). (C) CTLA4-FasL effects on the expression of apoptotic and anti-apoptotic proteins in B cell lymphoma cell lines (left) and RCC (right). Raji and A498 cell lines were incubated with indicated concentrations of CTLA4-FasL, sFasL, CTLA4-Ig or the combination of the later two for 2 h. Whole cell lysates were analyzed by Western blot. These are representative results of the three independent experiments. (D) Effect of B7 blockade on CTLA-FasL's effect on pro and anti apoptotic signals - Raji cell lines were incubated for 1 h with 1 μg/ml of anti CD80, anti CD86 or both prior to the addition of CTLA4-FasL (50 ng/ml). Cells were collected after 2 h. Whole cell lysates were analyzed by Western blot. (E) CTLA4-FasL effect on NFκB pathway - A498 (lower panel) and Raji (upper panel) cell lines were incubated with indicated concentrations of CTLA4-FasL, sFasL, CTLA4-Ig or the combination of the later two for 2 h (Raji) or 6 h (A498). Whole cell lysates were analyzed by Western blot.