Fig. 5: Auranofin induces cuproptosis-like cell death by thiol exchange.
a mass spectrometry identification of the products after thiol exchange during the 0.5-hour incubation of auranofin (100 μM) and lipoic acid (1 mM) in the cell lysate (500 million cells to 400 μL lysates in ddH2O). Created in BioRender. Xiong, X. (2025) https://BioRender.com/i289oxu. b analysis of the cuproptosis markers (LIAS and FDX1) in HCT116 cells after a 24-hour auranofin treatment in the presence of different concentrations of FBS. The samples derive from the same experiment but different gels for anti-LIAS and anti-FDX1 antibodies were processed in parallel. c qRT-PCR analysis of the mRNA levels of indicated genes after a 12-hour treatment with 4 μM auranofin. Data are shown as mean ± s. d. of four independent experiments. Significance was calculated by unpaired, two-tailed t-test. Significance was defined as p < 0.05. d DLAT aggregation measurement by reduced and non-reduced blots under indicated doses of auranofin in 24-hour treatments. A red arrow marked the band of aggregated DLAT proteins. e measuring the lipoylation of DLAT protein under indicated doses of auranofin in 24-hour treatments. The DLAT protein in the treated cells was enriched by its cognate antibody before the detection of lipoylated DLAT by the anti-lipoylation antibody. For c–e, treatments were performed in RPMI + 10% FBS. f the molecular markers of cuproptosis under auranofin (5 μM) mono or combined treatments (TGTA, 20 μM; pantethine, 100 μM) in RPMI + 30% FBS. The samples derive from the same experiment, but different gels for anti-LIAS and anti-FDX1 antibodies were processed in parallel. For b, d–f, each blot is a representative of three biologically independent experiments. g cell viability assay comparing the boost effect of 2 μM elesclomol (ES) towards copper chloride (2 μM) and auranofin (2 μM) on HCT116 after a 24-hour incubation. Data are shown as mean ± s. d. of six independent experiments. Significance was calculated by unpaired, two-tailed t-test. h Effects of tetrathiomolybdate (50 μM) on cytotoxicity of copper chloride and auranofin treatments on HCT116 after a 24-hour incubation. The fold change of IC50 to that of the control group was shown. For g, h, treatments were performed in RPMI + 10% FBS, and data were presented as mean ± s. d. from 3 biologically independent replicates. Significance was calculated by an unpaired, two-tailed t-test. Significance was defined as p < 0.05. Source data are provided as a Source Data file.
