Figure S2.

TRX-COBI acts through ferrous iron–mediated payload (COBI) release and resultant MEK inhibition. (A) Representative images of H&E (40×) showing the epidermal layer of mouse tail skin after treatment with vehicle or binimetinib for 20 d. The representative image of vehicle is the same as in Fig. 5 E. Scale bar: 20 µm. (B) Sensitivity of pancreatic and breast cancer cells to TRX-COBI expressed as a GI₅₀ ratio, defined as the GI₅₀ COBI divided by the GI₅₀ of TRX-COBI. Cells were treated for 5 d. (C) Quantification of intracellular Fe²⁺ levels measured by SiRhoNox in MiaPaca2 and Capan-1 cells with STEAP3 knockdown and iron chelator DFO treatment. (D and E) Phospho-ERK/total ERK levels in MiaPaca2 and Capan-1 cells with STEAP3 knockdown or after 24 h of DFO (iron chelator) treatment. Cells were treated with the indicated concentration of TRX-COBI for 2 h before lysis. Error bars indicate ±SD. (F) ROS production in MiaPaca2 cells assessed by flow cytometry after cells were incubated with vehicle (DMSO), COBI (100 nM for 6 h), TRX-COBI (100 nM for 6 h), or H₂O₂ (50 μM for 1 h). Quantification for the fluorescent intensity. Error bars represent mean ± SEM, analyzed by a two-sample t-test. **, P < 0.01. (G) Lipid peroxidation in MiaPaca2 cells as assessed by flow cytometry using C11 BODIPY after cells were incubated with either vehicle (DMSO), COBI (100 nM, 10 h), TRX-COBI (100 nM, 10 h), or the ferroptosis inducer erastin (10 μM for 10 h). Quantification for the fluorescent intensity. Error bars represent mean ± SEM, analyzed by a two-sample t-test. **, P < 0.01. (H) Cell viability was measured after 24 h incubation in MiaPaca2 and PANC02.03 cells. Cells were incubated with single-agent COBI (100 nM), or single-agent TRX-COBI (100 nM) or erastin (10 µM), or cotreatments including COBI + DFO (100 µM), COBI + ferrostatin-1 (1 μM), TRX-COBI + DFO (100 µM), TRX-COBI + ferrostatin-1 (1 μM), or erastin + ferrostatin-1 (1 μM). Error bars represent mean ± SEM, analyzed by a two-sample t-test. **, P < 0.01. Each panel in F and G is representative of three independent experiments.