Fig. 5: F. nucleatum induced ferroptosis in the colonic epithelial cells.

A CCK-8 Assay Kit determined the cell survival (%) of CCD-841(n = 6). B LDH Assay Kit determined the LDH releasing (%) of CCD-841(n = 6). C MDA Assay Kit measured the MDA levels of CCD-841(n = 6). D GSH and GSSG Assay Kit measured the relative GSH/ GSSG ratio (GSH%) of CCD-841(n = 6). E Western blotting analysis of GPX4, FTH1, and ACSL4 and quantification. ɑ-tubulin was used as the loading control (n = 3). F RT-qPCR measured the relative mRNA level of GPX4, FTH1, and ACSL4 (n = 6). G FeRhoNox-1 fluorescence staining for detecting Fe²⁺ of CCD-841 and quantification were shown on the right (n = 6). H Immunohistochemical staining for GPX4, FTH1, and ACSL4 (scale: 200 μm, left; 100 μm, right). The average optical density of GPX4, FTH1, and ACSL4 were shown on the right (n = 6). After the introduction of Fer-1 and DFO, I C11-BODIPY staining assessed the ROS level of CCD-841 and quantification (n = 3). Oxidation state BODIPY signal (green), reduction state signal (red). J JC-1 fluorescence staining detected the mitochondrial membrane potential (MMP) of CCD-841 and quantification. When MMP is high, JC-1 aggregates in the matrix of mitochondria and produces red fluorescence; at lower MMP, green fluorescence is produced (n = 3). Each dot indicated an individual mouse. All data represent the mean ± SEM. *P < 0.05, **p < 0.01, ***P < 0.001, ****p < 0.0001, by one-way or two-way ANOVA, followed by a post hoc test.