Fig. 3
METTL3 induced the H3K27 trimethylation of SLC7A11 promoter via suppression of KDM6B. A ChIP-qPCR assay was performed to examine H3K27me3, H3K4me3 and H3K27ac binding to the SLC7A11 promoter in HeLa cells. B After treating HeLa (left) and MDA-MB-231 cells (right) with 2 μM erastin, ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11. C ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11 after transfecting with siRNAs of KDM6B for 24 h in HeLa WT cells. D ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11 after transfecting with siRNAs of KDM6B for 24 h in HeLa WT and METTL3 KD cells. E ChIP-qPCR analysis was performed to examine the binding of H3K27me3 at the promoter region of SLC7A11 after transfecting with siRNAs of KDM6B for 24 h in MDA-MB-231 sh-NC and sh-METTL3 cells. F SLC7A11 mRNA was detected after transfecting with siRNAs of KDM6B for 24 h in HeLa cells by qRT-PCR. G SLC7A11 and KDM6B proteins were detected after transfecting with siRNAs of KDM6B for 48 h in HeLa cells by western blot (left) and quantitatively analyzed (right). H H3K27me3 and KDM6B proteins were detected by western blot (left) and quantitatively analyzed (right) in HeLa WT and METTL3 KD cells. I One m6A peak was enriched in KDM6B from m6A RIP-seq data. J m6A RIP-qPCR analysis of KDM6B mRNA in HeLa WT or METTL3 KD cells. K m6A RIP-qPCR analysis of KDM6B mRNA in MDA-MB-231 sh-NC or sh-METTL3 cells. L After treatment with Act-D for the indicated times, the mature mRNA levels of KDM6B were checked in WT and METTL3 KD HeLa cells. M YTHDF2 RIP-qPCR analysis of KDM6B mRNA in WT or METTL3 KD HeLa cells. N KDM6B protein was detected after transfecting with vector or YTHDF2 plasmid for 48 h in HeLa cells by western blot analysis (right), and mRNA by qRT-PCR (left). O After transfecting with control or YTHDF2 plasmid for 24 h, Act-D was added for the indicated times; furthermore the mature mRNA levels of KDM6B were checked in HeLa cells. Data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant, by Student’s t test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison