Fig 2: Auranofin (AU)-induced ROS: Cytotoxic and molecular ROS-dependent effects in U87/EGFR isogenic cell lines. (A) Fold change of relative fluorescence units (RFU) of ROS intracellular levels in U87MG, U87/EGFRwt, and U87/EGFRvIII cells using a microplate reader (AU, 3 µM, 24 h). Error bars represent the standard error of the mean (SEM) (* p < 0.05, *** p < 0.001). (B) MTT assay showing the effect of AU alone or in combination with N-acetylcysteine (NAC) on cell vitality (left); bars show the mean ± SEM (*** p < 0.001). NAC protection effects on vitality relative to AU-treated cells (right); bars show the mean ± SEM (* p < 0.05, ** p < 0.01, and *** p < 0.001). (C) Phase contrast imaging of cell morphology upon treatment with AU or in combination with NAC. (D) Colony-formation assay plates representative of Figure 2G; (6× magnification). (E) Clonogenic assay showing surviving fraction (Log Scale) following treatment with AU 0.5 μM alone or in combination with NAC, 2 mM for 9–11 days; NAC protective effect against decreased clonogenic survival is expressed as a percentage relative to AU-treated cells. Bars show the mean ± SEM (*** p < 0.001). Graphs represent mean values ± SEM from at least three independent experiments in triplicate. (F) Western blotting analysis showing the impact of auranofin alone (3 µM, 24 h) or in presence of NAC 2 mM on phosphorylated EGFR, total EGFR, (G), NRF2, TrxR1 (H), PARP-1 cleavage, and protein polyubiquitination in the three cell lines. DMSO was used as a vehicle control and actin as a loading control. “Shorter exposure” refers to a reduced time exposure to avoid signal saturation for U87/EGFRvIII.

Fig 3: Auranofin (AU) cytotoxicity in U87/EGFR isogenic GBM cell lines. (A) Western blot analysis of phosphorylated-EGFR Tyr1068 (p-EGFR), total EGFR, and TrxR1 basal expression levels in U87MG cells, U87/EGFRwt, and U87/EGFRvIII GBM cell lines. β-actin was used as a loading control. (B) TrXR activity after treatment with 0.5 μM AU for 24 h compared to DMSO control. Error bars represent the standard error of the mean (SEM) (* p < 0.05, ** p < 0.01) (C) MTT assay showing the IC50s of the EGFR isogenic cell lines in cells treated with AU for 72 h. (D) Viability and (E) total cell count of cells treated with AU for 72 h; bar charts show the mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001). (F) Clonogenic assay showing colony-formation capacity at different concentrations of AU after 9–10 days incubation. (G) Microscopic images of colonies in control (0 µM) and abortive colonies (<50 cells) following treatment with AU at 1 µM. (H) Survival fraction (Log scale) of U87MG, U87/EGFRwt, and U87/EGFRvIII cells; each data point shows the mean ± SEM; significant difference between U87/EGFRwt and U87/EGFRvIII at 0.5 µM AU (* p < 0.05). (I) Residual cytotoxicity of AU (Log scale): Colony formation capacity after treatment with AU for 72 h, followed by plating viable cells in drug-free media for 9–10 days. Each data point shows the mean ± SEM; significant difference between U87/EGFRwt and U87/EGFRvIII at 3.0 µM AU (* p < 0.05, ** p < 0.01); significant difference between U87/EGFRwt and U87MG at 3.0 µM AU (*** p < 0.001). Graphs represent the mean ± SEM from three independent experiments in triplicate.