Figure 2. FB₁ prevents radiation-induced MOMP in HeLa cells.

(A) FB₁ blocks mitochondrial ceramide generation. HeLa cells were irradiated (IR) and treated with 15 µM FB₁ 20 h post-irradiation. Ceramide in isolated mitochondria was quantified by diacylglycerol kinase assay at 36 h post-irradiation. Data (mean±SEM) are from 2 experiments performed in triplicate. *, p<0.05 vs. control; **, p<0.01 vs. irradiated. (B) FB₁ prevents radiation-induced Bax insertion into the MOM. Alkali-resistant mitochondrial fractions containing inserted Bax were isolated after 34 h from HeLa cells irradiated with 20 Gy and treated with 25 µM FB₁ or 75 nM ISP-1 at 20 h post-irradiation. COXII was used as mitochondrial loading control. Data are from 1 of 4 studies. (C) FB₁ blocks radiation-induced Bax oligomerization. At 34 h post-irradiation, mitochondrial proteins from HeLa cells treated as in (B) were separated by gel filtration. Data are from 2 studies. (D) FB₁ attenuates radiation-induced cytochrome c release in HeLa cells and BAEC. HeLa cells were irradiated with 10 Gy, and 15 µM FB₁ was added 20 h post-irradiation. BAEC cells were treated with 25 µM FB₁ 1 h before irradiation with 5 Gy. 36 h (HeLa) and 12 h (BAEC) post-irradiation, cytosolic fractions were analyzed by immunoblotting using mouse monoclonal anti-Cyt. c and mouse monoclonal anti-tubulin antibodies. Data are from 1 of 2 studies in HeLa and BAEC each. (E) FB₁ attenuates radiation-induced caspase activity. FB₁ was added to cells 20 h after 10 Gy and caspase activity measured at 36 h post-irradiation using the fluorogenic caspase substrate Z-DEVD-AFC. Data (mean±SEM) are from 1 of 2 investigations performed in triplicate.