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. Author manuscript; available in PMC: 2021 Dec 3.
Published in final edited form as: Mol Cell. 2020 Oct 30;80(5):828–844.e6. doi: 10.1016/j.molcel.2020.10.010

Figure 7. Combined Loss of NRF2 and Inhibition of GPX4 Leads to Death of both Inner and Outer Cells of Spheroids.

Figure 7.

(A and B) Representative confocal images of the indicated spheroids from three independent experiments. Spheroids were treated with 10 μM ML210 and 1 μg/ml doxycycline for the indicated time periods. (C–E) Total area of cells/spheroids relative to ML210-untreated controls in the indicated cells treated with ML210 for the last three days. A549 and H1437 cells were cultured in 2D for four days or 3D for 12 days in the presence of either 1 μg/ml doxycycline (C) or both 1 μg/ml doxycycline and 3 μM Fer-1 (D and E). H520 and SK-MES-1 cells were cultured in either 2D for four days or 3D for 15 days in the presence of 1 μg/ml doxycycline. (F) Representative C11-Bodipy ratiometric images of the indicated day-10 A549 spheroids upon treatment with 1 μM ML210 for the last three days from two independent experiments. (G) Quantification of C11-Bodipy ratio in the indicated day-10 A549 spheroids treated with or without 1 μM ML210 for the last three days. The data for both ML210-untreated spheroids and ML210-treated spheroids with shControl are from Figures 5I, 5J, and S4I as these experiments were performed concurrently as the experiment presented in Figure 7G. Data shown as mean ± SEM (n = 8–20). One-way ANOVA was used to determine statistical significance. *p < 0.05 and ***p < 0.001. In (C), (D), and (E), data shown as mean ± SD from four independent experiments, and two-way ANOVA was used to determine statistical significance (*p < 0.05, **p < 0.01, and ***p < 0.001 compared to shControl). Scale bar represents 100 μm.