IRP1 and ARID3A reduce the chromatin accessibility of the CYGB promoter region and inhibit CYGB expression. (IMAGE)
Caption
(A) The intersection of down-regulated peaks in ATAC sequencing, up-regulated genes in RNA sequencing, and the Ferroptosis driver database. (B) The binding peak of ARID3A and chromatin accessibility regulated by IRP1 in the promoter region of CYGB were visualized by an integrative genomics viewer (IGV). (C) CYGB mRNA expression level in ARID3A-knockdown Patu-8988 cells was detected by quantitative reverse transcription PCR (∗∗P < 0.01, ∗∗∗P < 0.001). (D) CYGB mRNA expression level in ARID3A-overexpressing PANC-1 cells was detected by quantitative reverse transcription PCR (∗P < 0.05, ∗∗∗P < 0.001). (E) ARID3A, IRP1, and CYGB protein expression levels were detected in PANC-1 and Patu-8898 cells by Western blotting to verify the relationship between ARID3A, IRP1, and CYGB under the condition of iron overload. (F) CYGB expression level was detected in Patu-8898 cells by Western blotting to verify the relationship between ARID3A, IRP1, and CYGB under the condition of iron overload (∗∗∗P < 0.001). (G) CYGB expression level was detected in PANC-1 cells by Western blotting to verify the relationship between ARID3A, IRP1, and CYGB under the condition of iron overload (∗P < 0.05, ∗∗∗P < 0.001). (H, I) Chromatin immunoprecipitation assay performed with ARID3A antibody followed by detection of CYGB promoter through quantitative reverse transcription PCR in Patu-8988 and PANC-1 cells (normal condition, erastin added, hemin added; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). (J) DCFH-DA staining (green) was used to detect ROS generation in Patu-8988 cells transfected with control, siRNAs targeting CYGB. (K) The oxygen consumption rates of Patu-8988 cells were shown as a bar chart (∗∗∗P < 0.001). (L) DCFH-DA staining (green) was used to detect ROS generation in PANC-1 cells transfected with the vector, overexpression plasmid targeting CYGB. (M) The oxygen consumption rates of PANC-1 cells were shown as a bar chart (∗∗∗P < 0.001). (N) MDA levels were detected in Patu-8988 cells transfected with control, siRNAs targeting CYGB (∗∗∗P < 0.001). (O) MDA levels were detected in PANC-1 cells transfected with the vector, overexpression plasmid targeting CYGB (∗∗∗P < 0.001). MDA, malondialdehyde; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; ROS, reactive oxygen species.
Credit
Zeru Li, Cheng Qin, Bangbo Zhao, Tianyu Li, Yutong Zhao, Lirui Huang, Haoyu Shi, Yiping Xie, Yutong Yan, Xiangyu Zhang, Weibin Wang
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