Extended Data Fig. 3: CGI1746 acts as a new class of ferroptosis inhibitor.
From: CGI1746 targets σ1R to modulate ferroptosis through mitochondria-associated membranes
a. Immunoblotting for GPX4, SLC7A11, DHODH, TFRC and ACSL4 protein levels in MDA-MB-468 cells subjected to RSL3 treatment (1 μM) for indicated hours, with or without CGI1746 pre-treatment (10 μM). b-d. MDA-MB-468 cells were treated by 10 μM CGI1746 and/or 100 μM DFO, then labeled with FerroOrange to detect the cellular labile iron pool by confocal microscope (b and c) or flow cytometry (d). DFO was utilized as a positive control to reduce the cellular labile iron pool. The presented images are representatives (b, Scale bars, 20 μm). The labile iron levels in b were quantified in c. e. The effect of CGI1746 on chelating labile iron up to 200 μM was determined by Ferrozine–iron chelation assay, using DFO (100 μM) as a positive control. f. GSH abundance was measured by GSH-Glo™ Glutathione Assay in NCI-H1299 cells treated with erastin for 18 hours in the absence or presence of CGI1746 (10 μM). g. GPX4 degradation was analyzed using western blot in NCI-H1299 cells treated with erastin (5 μM) for indicated hours with or without CGI1746 (10 μM) pre-treatment. h. The capacity of antioxidant activity of CGI1746 in DPPH assay. Viability data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. For bar graphs c and e, data represent mean ± s.d. of n = 3 replicates from one representative of three independent experiments. Micrograph data show one representative out of three replicates from one representative of three independent experiments. Flow cytometry data show one representative out of two replicates from one representative of three independent experiments. Western blot data show one representative out of three independent experiments. ****P < 0.0001 and ns, not significant. One-way ANOVA (GraphPad Prism 9.5.1).
