Fig. 2: FOXO3 activation does not protect against ferroptosis through contact inhibition.
From: Activation of a FOXO3-induced cell cycle arrest regulates ferroptosis
A Characterization of hTERT-RPE-1 F3A3 p27-/- cell line after FOXO3 activation. Cells were treated with doxycycline for 24 or 48 h, and expression of FOXO targets was analyzed by Western blot. Total cell lysate of wild-type hTERT-RPE-1 F3A3 cells was used to determine band height of p27. (n = 3, one-way ANOVA). B Cell cycle profile of hTERT-RPE-1 F3A3 WT and hTERT-RPE-1 F3A3 p27-/- cells after either untreated or treated with doxycycline for 24 h and analyzed by FACS. (n = 3, one-way ANOVA). C hTERT-RPE-1 F3A3 WT and hTERT-RPE-1 F3A3 p27-/- cells were either left untreated or treated with doxycycline for 24 h, followed by an 8 h treatment with either a vehicle or 200 nM RSL3. The difference in cell viability between doxycycline-treated and untreated conditions was defined as the rescue percentage (Viabilitydox−ViabilityCtrl). (n = 3, unpaired t test with significant differences in rescue percentage between WT and p27-/- cells). D hTERT-RPE-1 cells were plated at spare or dense density, followed by an 8 h treatment with either vehicle or 200 nM RSL3 and analyzed by FACS. (n = 3, one-way ANOVA). E HTERT-RPE-1 cells were plated at indicated seeding density. 200 nM RSL3 and 100 µM Fe2+ were used to induce lipid peroxidation. (n = 3, one-way ANOVA). F hTERT-RPE-1 F3A3 cells were plated at sparse density. Cells were either left untreated or treated with doxycycline for 24 h and thereafter treated for 8 h with indicated dose of RSL3. (n = 3, unpaired t test with significant differences from dose 200 nM). G hTERT-RPE-1 p27-/- cells were plated at sparse or dense confluency, followed by an 8 h treatment with either vehicle or 200 nM RSL3 and analyzed by flow cytometry. (n = 3, one-way ANOVA).
