Figure 6.

Hs578T cells were seeded in six-well plates at 0.1 × 10⁶ cells in GM followed by 24 h in SFM. Cells were pre-dosed for 24 h with IGFBP-3 (100 ng/ml) prior to redosing ± an apoptotic dose of antimycin A (A). Graph represents cell surface ceramide as assessed by flow cytometry and is the mean ± SEM of three experiments. Cells were pre-dosed with IGFBP-3 for 24 h ± a ceramide blocking antibody (1 μg/ml) prior to redosing ± an apoptotic dose of antimycin A (B). Graph represents changes in cell death and shows the mean ± SEM of three experiments each repeated in triplicate. (C) Shows phase contrast photomicrographs (magnification ×100) of Hs578T cells following treatment described in (B), which are representative of experiments repeated three times. Cells were pre-dosed for 24 h with IGFBP-3 (100 ng/ml) ± DMS (0.5 μM) and then re-dosed ± an apoptotic dose of C2-ceramide (D). The graph represents changes in cell death and shows the mean ± SEM of three experiments each repeated in triplicate.