Romo1-induced membrane permeabilization. (A–E) Romo1-induced liposome permeabilization. CF-LUVs were incubated with the indicated concentrations of Romo1, and liposome permeabilization was monitored by a spectrophotometric assay (A). (B) CF release was quantified at 10 min after Romo1 treatment. (C) Illustration of deletion mutants of Romo1. (D and E) CF-LUVs were incubated with Romo1 or the indicated deletion mutants (1 µM) for 10 min, after which liposome permeabilization was analyzed. Alamethicin (Ala.) was used as a positive control. (F–K) Romo1-induced mitochondrial depolarization. (F) The purity of isolated mouse liver mitochondria was examined by flow cytometry using the mitochondrial-specific marker MitoTracker green FM. (G and H) Mitochondrial targeting of Romo1 was analyzed by flow cytometry with 0.2 µM TAMRA-Romo1 (G) and confirmed by confocal microscopy (H). Bar, 10 µm. (I and J) Isolated liver mitochondria were treated with the indicated concentrations of Romo1 for 5 min and then stained with the Δψm indicator JC-1, after which they were analyzed by flow cytometry (I) and spectrophotometric assay (J). (K) Romo1 or its deletion mutants (1 µM) were added to isolated liver mitochondria for 5 min followed by staining with JC-1, after which Δψm was quantified by spectrophotometric assay. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was used as a positive control. (L–N) CF-LUVs were incubated with the indicated deletion or point mutants of Romo1 (1 µM) for 10 min, and liposome permeabilization was quantified by a spectrophotometric assay. Data represent means ± SD of three independent experiments. ***, P ≤ 0.001 by two-way ANOVA. Cont., control.