Journal: Cell Death Discovery

Article Title: Rafoxanide disrupts mitochondrial homeostasis through VDAC1 modulation in colorectal cancer cells

doi: 10.1038/s41420-026-02986-3

Figure Lengend Snippet: Representative western blots showing VDAC1 monomeric and dimeric forms, together with β-actin, in HCT116 cells pre-treated or not with the specific VDAC1 inhibitor VBIT12 (40 µM) for 2 h and then stimulated with rafoxanide (RFX, 2.5 μM) or DMSO (vehicle) for 5, 15, 30, and 60 min. One of 3 independent experiments in which similar results were obtained is shown.

Article Snippet: HCT116 cells were pre-treated for 2 h with the VDAC1 inhibitor VBIT12 (40 μM, #S8936, Selleck Chemicals LLC, Houston, Texas) to block VDAC1 oligomerization or for 1 h with N-acetylcysteine (NAC, 1 mM, #A0150000, Sigma Aldrich) to decrease reactive oxygen species (ROS) level.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: Rafoxanide disrupts mitochondrial homeostasis through VDAC1 modulation in colorectal cancer cells

doi: 10.1038/s41420-026-02986-3

Figure Lengend Snippet: A Detection of total cellular reactive oxygen species (ROS) by fluorescence intensity measurement in HCT116 cells pre-treated or not with the ROS scavenger NAC (1 mM) for 1 h and then incubated with fluorescence probe DCFDA. After 30 min, cells were washed and stimulated with rafoxanide (RFX, 2.5 μM) or DMSO (vehicle). Fluorescence intensity was measured at the baseline and after 5, 15, 30, and 60 min upon stimulation. Differences among groups were compared using one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test (* P ≤ 0.05, ** P ≤ 0.01). One of 3 independent experiments where similar results were obtained. B Representative western blots showing VDAC1 monomeric and dimeric forms, together with β-actin, in HCT116 cells pre-treated or not with the ROS scavenger NAC (1 mM) for 1 h and then stimulated with rafoxanide (RFX, 2.5 μM) or DMSO (vehicle) for 1 h. One of 3 independent experiments in which similar results were obtained is shown. C Left panel : representative flow cytometry dot plots showing JC-1 fluorescence in HCT116 cells treated as indicated in panel ( B ). JC-1 aggregates indicate polarized mitochondria, whereas JC-1 monomers indicate mitochondrial membrane depolarization. Numbers indicate the percentage of cells in the designated quadrants. Right panel : quantification of the fraction of cells with depolarized mitochondria. Values are mean ± SEM of 3 independent experiments. Differences among groups were compared using one-way ANOVA followed by Tukey’s post hoc test (** P ≤ 0.01, *** P ≤ 0.001). D , E Complex I ( D ) and complex III ( E ) activity assays were performed spectrophotometrically in HCT116 cells stimulated with rafoxanide (RFX, 2.5 μM) or DMSO (vehicle) for 5 and 15 min. Values are mean ± SEM of 3 independent experiments. Differences among groups were compared using one-way ANOVA followed by Tukey’s post hoc test (* P ≤ 0.05, ** P ≤ 0.01). F Calcium mobilization was measured by flow cytometry in HCT116 cells stained with the 520-AM dye for 30 min, washed, and stimulated with rafoxanide (RFX, 2.5 μM), DMSO (vehicle), or ionomycin (positive control). Samples were acquired by flow cytometry at the baseline and immediately after stimulation. One of 3 independent experiments in which similar results were obtained is shown.

Article Snippet: HCT116 cells were pre-treated for 2 h with the VDAC1 inhibitor VBIT12 (40 μM, #S8936, Selleck Chemicals LLC, Houston, Texas) to block VDAC1 oligomerization or for 1 h with N-acetylcysteine (NAC, 1 mM, #A0150000, Sigma Aldrich) to decrease reactive oxygen species (ROS) level.

Techniques: Fluorescence, Incubation, Western Blot, Flow Cytometry, Membrane, Activity Assay, Staining, Positive Control