Journal: bioRxiv

Article Title: Assembling Ruthenium Complexes to Form Ruthenosome Unleashing Ferritinophagy-mediated Tumor Suppression

doi: 10.1101/2024.10.22.619580

Figure Lengend Snippet: (a) Fluorescent images of HCT116 cells with and without the treatments of Ru-complexes (300 nM, 24 hours) stained with FeRhoNox-1. Scale bars represent 20 μm. (b) Confocal fluorescence images of HCT116 cells incubated with C11-BODIPY after treatment with Ru-complexes at a concentration of 300 nM for 24 hours. The fluorescence transition from red to green indicated significant mitochondrial damage. Scale bars represent 20 μm. (c) Cellular component (CC) of GO enrichment analysis of differentially expressed genes in HCT116 cells treated with RuOA2 (300 nM) for 24 hours compared with the control. Bubble diagram was made based on the genes shown the top 30 CCs with high gene number. Bubble size represents the number of genes, while color represents the rich factor of gene expression. (d) Immunoblots for GPX4, NCOA4, FTH1, ATG5, ATG7, and LC3B I/II expression in HCT116 cells upon the treatment of Ru-complexes at a concentration of 300 nM for 24 hours. β -actin serves as the loading control. Immunoblots repeated in triplicate with similar results. (e) Relative intracellular GSH level in HCT116 cells upon treatment with Ru-trisBP@DOPC, Ru-trisBP@OA, and RuOA2 at a concentration of 300 nM for 24 hours. Mean ± SD (n=3). (f) The ferrous ion content in HCT116 cells transfected with control vector or FTH1-overexpression encoding plasmids with or without the treatment of 300 nM RuOA2 for 24 hours. Mean ± SD (n=3). (g) Viability of HCT116 cells transfected with control vector or FTH1-overexpression encoding plasmids with or without the treatment of 300 nM RuOA2 for 24 hours. Mean ± SD (n=6). (h) TEM images (false color) of RuOA2 (1μM, 4 hours) treated HCT116 cells. The mitochondria are highlighted in pink, the phagophores are highlighted in light blue, the autophagosomes are highlighted in green, and the autolysosomes is highlighted in yellow. Scale bars represent 500 nm. (i) HCT116 cell viability upon the treatment of RuOA2 at a concentration of 400 nM for 24 hours after the pre-treatment of different PCD inhibitors. Mean ± SD (n=8). ns: no significant, ***p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies included: anti-β-Actin (66009-1-Ig, Proteintech, 1:5000), anti-LC3B I/II (ET1701-65, HuaBio, 1:1000), anti-ATG5 (ET1611-38, HuaBio, 1:1000), anti-ATG7 (ET1610-53, HuaBio, 1:1000), anti-NCOA4 (ER62707, HuaBio, 1:2000); anti-FTH1 (T55648, Abmart, 1:1000); anti-GPX4 (T56959S, Abmart, 1:1000).

Techniques: Staining, Fluorescence, Incubation, Concentration Assay, Control, Expressing, Western Blot, Transfection, Plasmid Preparation, Over Expression