Figure 7
Assessment of both mitochondrial and cellular toxicity.
(a,b) Rat liver mitochondria (1 mg protein ml−1) were incubated with compound for 10 minutes in the presence of either glutamate (10 mM) and malate (2 mM) or succinate (10 mM) and rotenone (1 μM). ADP (5 mM) was added and incubated for 45 minutes. Mitochondria were lysed and total ATP quantified by luminescence using CellTiter Glo reagent (Promega, Madison, WI). No effect of either ER-000444793, CsA or SfA was observed under either respiratory substrate condition. Results are expressed as % inhibition of ATP, synthesis normalised to DMSO (0% inhibition) and antimycin A (2.5 μM) plus oligomycin A (2.5 μg ml−1; 100% inhibition). Data are expressed as means (± s.d.) of at least three independent experiments. (c) Assessment of immunosuppression and cellular toxicity in Jurkat NFAT-reporter cells. Jurkat cells were incubated with compound for 45 minutes prior to the addition of ionomycin (0.5 μM) and PMA (50 nM). NFAT reporter activity was assessed after 20 hours treatment with either ER-000444793, CsA or SfA and quantified by luminescence using Bright Glo reagent (Promega, Madison, WI). Data are expressed as % inhibition, normalised to DMSO (0% inhibition) and CsA (5 μM; 100% inhibition). (d) Toxicity of ionomycin/PMA treatment was assessed in Jurkat cells using Alamar blue after 20 hours incubation. No significant effect was observed with treatment. Data are expressed as fluorescence intensity and statistical significance calculated using paired two-tailed student’s t-test using GraphPad Prism (NS P > 0.05). (e) Jurkat cells were incubated with compound for 20 hours and toxicity assessed using Alamar Blue. Data are expressed as fluorescence intensity and compared using multiple t-tests comparing treatments to DMSO, corrected for multiple comparisons using Holm-Sidak method, P < 0.05 defined as being statistically significant. All data are expressed as means (± s.d.) of three independent experiments. Abbreviations: CsA; cyclosporin A, SfA; sanglifehrin A, Cmp; compound, O.D; optical density, NS; not significant, PMA; phorbol 12-myristate.
