Figure 7.

FeCl₂ does not interfere with the activities of sterubin. (A) FeCl₂ does not alter the protective effect of sterubin against glutamate toxicity. HT22 cells were treated with 2.5 mM glutamate in the presence of 5 µM sterubin and/or FeCl₂ 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. (B) FeCl₂ does not affect the maintenance of GSH levels by sterubin. HT22 cells were treated with 2.5 mM glutamate in the presence of 5 µM sterubin and/or FeCl₂ at the indicated concentrations. Cells were harvested after 24 h and total GSH was measured. The results are the average of 5–6 independent experiments. (C) FeCl₂ only slightly impairs the protective effect of sterubin against RSL3 toxicity. HT22 cells were treated with 100 nM RSL3 in the presence of 5 µM sterubin and/or FeCl₂ 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. (D–E) FeCl₂ does not reduce the anti-inflammatory effects of sterubin. BV-2 cells were treated overnight with 25 ng/mL LPS in the presence of 5 µM sterubin alone or with the addition of the indicated concentrations of FeCl₂. Cell culture supernatants were cleared and assayed for NO by the Griess assay or IL-6 using a specific ELISA. Results are presented as the percent (%) of the value obtained with LPS alone which was set at 100%. ** indicates p < 0.01 relative to sterubin + RSL3 alone. ^ indicates p < 0.05 and ^^^ indicates p < 0.001 relative to insult alone. (F) Quantitation of results from four independent experiments identical to (G). (G) Representative Western blots for Nrf2, ATF4 and actin of nuclear extracts from BV-2 cells treated with 25 ng/mL LPS alone or in the presence of 5 µM sterubin ± the indicated concentrations of FeCl₂ or CuCl₂ for 4 h.