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. 2021 Mar 24;11(12):5650–5674. doi: 10.7150/thno.55482

Figure 7.

Figure 7

Reduction of YAP sensitizes ferroptosis by elevating iron via FTH1. (A) Relative labile iron was measured in WT and βTrCP-/- H1975 cells after treating with or without erastin (10 µM) for 24 h. (B) Relative labile iron was measured in GGG cells under the same treatment as that in Figure 2D. The labile iron was normalized between those treated with erastin and DMSO. (C) p-YAPS127, O-YAPT241 and total YAP were measured by IB in H1975 cells treated with erastin (10 µM) for indicated hours. (D-F) Relative labile iron (D), lipid ROS generation (E) and cell death (F) were measured in LUAD cells treated with erastin (10 µM) for indicated hours. DMSO or DFO (A549, 2 µM; H358,15 µM; H1299, 5 µM; H1650, 18 µM; PC9, 8 µM; H1975, 25 µM) was added at 10 h post erastin treatment. (G) Correlation between induced cell death and remaining FTH1 level after erastin (10 µM) treatment for 24 h. Induced cell death and remaining FTH1 level was calculated as fold or percentage to the ones treated with DMSO. (H) FTH1 was knocked down in βTrCP-/- H1975 cells treating with erastin (10 µM) with or without Fer-1 (1 µM) for 24 h. Cell death was measured using SYTOX green staining followed by flow cytometry. (I-J) FTH1 protein (I) and mRNA (J) in H1975-based GGG cells pretreated with or without Dox (1 µg/ml) and EGCG (5 µM) for 24 h before further treating with erastin (10 µM) for indicated hours. The level of proteins was normalized to that of GAPDH, and the normalized level of proteins in erastin-treated 0 h cells was arbitrarily set to 1 (I). (K) Ferritinophagy boosts YAP binding to FTH1 promoter. H1975 cells with or without NCOA4 knockdown were treated with erastin (10 µM) for 4 h. Enrichments of YAP at TFCP2 motif within the FTH1 promoter were measured by ChIP-qPCR. (L-M) Dynamic YAP and TFCP2 binding to the FTH1 promoter triggered by erastin. WT or βTrCP-/- H1975-based GGG cells were under the same treatment as that in panel I. Enrichments of YAP (L) or TFCP2 (M) at TFCP2 motif within the FTH1 promoter were measured by ChIP-qPCR at indicated hours. The data are shown as the mean ± SD from three biological replicates (including IB). *P < 0.05, **P < 0.01 indicates statistical significance. Data in A, H, K were analyzed using a one-way ANOVA test. Data in B, J, L, M were analyzed using a two-way ANOVA test. Data in G were analyzed using the Spearman rank-correlation analysis.