Fig. 5: Methylation-originated ubiquinone synthesis is implicated in ROS generation during ferroptosis.

A Schematic of key proteins in SAM-based ubiquinone synthesis pathway. B HT1080 cells were transfected with the GFP-tagged Mito-pHyPer plasmid for 48 h, and then cultured as indicated for another 8 h. The mean fluorescence intensities (MFI) of Mito-pHyPer were quantified. C–E HT1080 cells were cultured as indicate to quantify the cellular levels of ROS (C) at the 10 h, the cell death levels (D) at the 14 h and the UQ levels (E) at the 8 h. F–I HT1080 cells were respectively transfected with siRNA targeting SLC25A26 (siSLC25A26-1/2 mixture), CoQ3 (siCoQ3-1/2 mixture) or CoQ5 (siCoQ5-1/2 mixture) for 48 h, and then cultured as indicated for another 8–14 h. (F) The UQ levels were detected at the 8 h. G, H The levels of cellular ROS and lipid ROS were determined at the 10 h. I The cell death levels were quantified at the 14 h. J HT1080 cells were transfected with SLC25A26 plasmid for 48 h, and then cultured in the medium ± cystine, ± methionine as indicated for another 14 h to quantify the cell death. The proteins of SLC25A26 and tubulin were determined by WB. K–M HT1080 cells were co-transfected with SLC25A26 and Mito-pHyper for 48 h, and then cultured in the medium ± cystine, ± methionine in the absence or presence of Mito-TEMPO as indicated for another 8–10 h. K, L The UQ levels and the MFI of Mito-pHyper were respectively quantified at the 8 h. M The lipid ROS levels were determined at the 10 h. CC, 0.4 mM; Met, 0.4 mM; SAM, 0.4 mM; FIDAS-5, 5 μM; ADOX, 20 μM; AA, 10 μM; MT, 5 μM. For B-M, n = 3 biological replicates, data were represented as mean ± SD with p values determined by one-way ANOVA test. Source data are provided as a Source Data file.