Skip to main content
. 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 5. NRF2 Controls Survival of Inner Spheroid Cells through Protection from Ferroptosis.

Figure 5.

(A) Representative confocal images of the indicated spheroids from three independent experiments. Spheroids were treated with or without 10 μM ML210 for the last three days. (B) Percentage of filled inner space from experiments described in (A) (n = 35–77). (C) Representative confocal images of the indicated spheroids from three independent experiments. Spheroids were treated with 1 μg/ml doxycycline and either vehicle or 3 μM Fer-1 for 12 days. (D) Percentage of filled inner space from experiments described in (C) (A549) and Figure S4G (H1437) (n = 11–77). (E) Representative confocal images of the indicated spheroids from three independent experiments. (F) Quantification of spheroid size from experiments described in (E) (n = 18–112). (G) Quantification of Ki67 signal relative to Fer-1-untreated spheroids with shControl from experiments described in (E) (n = 4–24). (H) H2O2 levels (arbitrary units) in the indicated day-10 spheroids from three independent experiments. (I) Representative C11-Bodipy ratiometric images of the indicated day-10 A549 spheroids from two independent experiments. Spheroids were treated with or without 1 μM ML210 for the last three days. (J) Quantification of C11-Bodipy ratio in inner region relative to outer region from experiments described in (I) (n = 12–20). (K) Fe2+ levels in the indicated day-10 spheroids from three independent experiments. (L) Immunoblot analysis of NRF2 and ACSL4 in H1437 spheroids. In (E–G), H1437 spheroids were treated with 1 μg/ml doxycycline and either vehicle or 3 μM Fer-1 throughout the 3D culture. In (H), (K), and (L), the spheroids were treated with 1 μg/ml doxycycline throughout the experiments. In (B), (H), and (J), unpaired two-tailed t-test was used to determine statistical significance. In (D), (F), and (G), one-way ANOVA was used to determine statistical significance. In (K), paired two-tailed t-test was used to determine statistical significance. *p < 0.05, **p < 0.01, and ***p < 0.001. All data shown as mean ± SEM. Scale bar represents 100 μm.