Figure 2

TCTP inhibits mitochondrial cytochrome c release and mitochondrial membrane depolarization in etoposide-induced cell death. (A) TCTP-induced inhibition of mitochondrial membrane perturbation. AdNull and adTCTP-infected cells (MOI, 10) were treated with 20 μM etoposide. Loss of mitochondrial membrane potential was measured using JC-1 dye by flow cytometry. The disrupter, CCCP was used as a positive control. JC-1 forms aggregates in the high mitochondrial membrane potential whereas the disrupted potential during apoptosis leads to form the JC-1 monomer. Loss of membrane potential was detected by measuring the shift of fluorescence from FL-2 (JC-1 aggregates, red fluorescence) to FL-1 (JC-1 monomers, green fluorescence) in FACS analysis. (B) TCTP-induced inhibition of mitochondrial cytochrome c release. AdGFP (G)- and adTCTP-GFP (T)-infected cells (MOI, 10) were treated with 20 μM etoposide. After fractionation of mitochondrial and cytosolic fractions, anti-cytochrome c antibodies were used for detecting its contents by western blot analysis. (C) Cytochrome c release was quantitated by expressing as RFU. AdGFP (G)- and adTCTP-GFP (T)-infected cells (MOI, 10) were treated with 20 μM etoposide. Following incubation with fluorescence-tagged cytochrome c-specific antibody, cells were subjected to FACS analysis. Data represent cytochrome c release relative to the control (mean ± S.D.) of two independent experiments.