Fig. 4: FSP1 is upregulated in KRASG12D-expressing cells in an NRF2-dependent manner.
From: Elevated FSP1 protects KRAS-mutated cells from ferroptosis during tumor initiation
a LsL-KRASG12D-inducible MEFs were treated for 72 h with or without tamoxifen (4OHT) [1 µg/ml] in 2% FCS before cells were starved overnight in 0.1% FCS and then refed with 2% FCS for the indicated timepoints. Cells were lyzed and subjected to protein analysis by Western blotting. b Levels of DUSP6, FSP1, NRF2, GCLC and HO-1 cDNA were quantified by qPCR in LsL-KRASG12D-inducible MEFs after 96 h or 120 h of tamoxifen (4OHT) treatment. Fold change relative to controls is shown. Means from MEF lines from 4–5 different embryos are shown. c Levels of KEAP1, FSP1, NRF2, GCLC and HO-1 cDNA were quantified by qPCR in Rasless MEFs expressing KRAS WT ± KEAP1 knockdowns for 72 h. Fold change relative to controls is shown. d siKEAP1 KRAS WT cells were treated after 48 h knockdown with DMSO, RSL3 [100 nM], ±iFSP1 [10 µM], ±Fer-1 [1 µM] for another 24 h. DRAQ7 [100 nM] was added to all wells to visualize dead cells. Images were acquired at ×10 magnification every 2 h using the IncuCyte S3 bioimaging platform. e Levels of NRF2, FSP1, GCLC and HO-1 cDNA were quantified by qPCR in Rasless MEFs expressing KRAS WT after cells were treated for 24 h with TBHQ [25 nM]. Fold change relative to controls is shown. f log2 mRNA expression data of A549 cells transfected with either siRNAs targeting NRF2 or GFP [33]. NRF2 mRNA expression is shown. g data as in f were analyzed for FSP1 (AIFM2) mRNA expression. Data are means ± SEM of three independent experiments in each individual cell line or representative images were applicable. Two-tailed t-test (b, c, e, f, g), Two-way ANOVA + Tukey’s multiple comparison test (d), ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Uncropped blots are provided as Original Data file.
