Fig. 5: CNMT K157 and E354 interacts with TFR1 to enhance iron overload for ferroptosis by PQS in macrophages.
A Proteome analysis to discover the potential direct ferroptosis-related target of PQS-enhanced CNMT. Schematic diagrams were created using BioRender.com. B Heatmap of significantly differentially expressed proteins (DEPs) in the shCNMT RAW264.7 macrophages+PQS compared with RAW264.7 macrophages based on proteome data. C A volcano plot of the DEPs between the RAW264.7 macrophages + PQS and shCNMT RAW264.7 macrophages+PQS based on proteome data was analysed. D Model of TFR1-dependent iron accumulation leading to lipid peroxidation and ferroptosis. Schematic diagrams were created using BioRender.com. E CO-IP assay of CNMT-Flag from the lysates of HEK293T cells co-transfected with TFR1-HA (n = 3). F Interaction model of the CNMT protein with the 67-amino acid TFR1 cytoplasmic region visualised using Chimera. G Interaction assay between CNMT and TFR1 using the Y2H system. The transformants were plated onto low-stringency Leu and Trp defect (left) and high-stringency Leu, Trp, His, and Ade defect (right) selection Synthetic Dextrose (SD) plates, respectively. CNMT* = CNMTK157G+E354G double mutation. H CCK-8 assay for cell viability in RAW264.7, shCNMT, shCNMT + siRNA-TFR1 double knockdown, and siTFR1 macrophages treated with PQS for 24 h. Data are presented as mean ± SD (n = 3, one-way ANOVA with Tukey’s multiple comparisons test). I Representative images of Phen Green fluorescence staining to label labile free iron in shCNMT + siTFR1 macrophages treated with or without PQS. Phen Green: green; Hoechst nucleus: blue. Scale bar = 20 µm. J Representative images of Phen Green fluorescence staining to label labile free iron in siTFR1 macrophages treated with or without PQS. Phen Green: green; Hoechst: blue. Scale bar = 20 µm. Source data are provided as a Source Data file.
