Figure 4.

FeCl₂ and CuCl₂ modulate the effects of fisetin on protection from multiple toxicities. FeCl₂ and CuCl₂ reduce fisetin-mediated protection against hydrogen peroxide (H₂O₂) (A) or t-butyl peroxide (tBOOH) (B) toxicity. HT22 cells were treated with 0.5 mM H₂O₂ or 2.5 µM tBOOH in the presence of 10 µM fisetin and/or FeCl₂ or CuCl₂ at the indicated concentrations. Cell survival was measured after 24 h with the MTT assay. The experiments were done in quadruplicate and the results are the average of 4 independent experiments. (C) FeCl₂ and CuCl₂ differentially affect fisetin-mediated protection against RSL3 toxicity. HT22 cells were treated with 100 nM RSL3 in the presence of 5 µM fisetin and/or FeCl₂ or CuCl₂ at the indicated concentrations. Cell survival was measured after 24 h with the MTT assay. The experiments were done in quadruplicate and the results are the average of 5 independent experiments. (A–C), * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001 relative to fisetin + toxin alone. (C), ^^ indicates p < 0.01 and ^^^ indicates p < 0.001 relative to RSL3 alone for both FeCl₂ and CuCl₂. (D) Cells were treated with 250 nM RSL3 alone or in the presence of 10 µM fisetin and 5 µM FeCl₂ or CuCl₂. After 4 h, ROS levels were determined using CM-H₂DCFDA as described in Materials and Methods. The treatments were done in sextuplicate and the results are the average of 4 independent experiments. ** p < 0.01, *** p < 0.001 relative to RSL3 alone. ^^^ p < 0.001 relative to RSL3 + FeCl₂ or CuCl₂ alone.