Fig 1: Kumatakenin reduced cellular iron levels by activating Eno-3 and inducing degradation of iron regulatory protein 1 (IRP1) mRNA. (A) Examination of Eno3 expression to verify the knockdown efficiency of siEno3. Effects of Eno3 knockdown on kumatakenin-exerted effects on (B) cellular iron levels and (C) lipid ROS in vitro. (D,E) Effects of kumatakenin on colonic IRP1 mRNA and protein expression in colitis mice. (F) IRP1 mRNA stability was determined in erastin-treated MODE-K cells by incubating with the transcription inhibitor actinomycin D (5 μM). (G) Effects of knockdown of Eno3 on IRP1 expression. (H) Examination of IRP1 expression to verify the knockdown efficiency of si-IRP1. Effects of Eno3 knockdown and IRP1 on cellular iron levels (I) and lipid ROS (J).
Fig 2: ACO1 is a mediator of exosomal miR‐148b‐3p delivery to macrophages. (a) RBPDB analysis was utilized to anticipate precise interaction between the miR‐148b‐3p sequence and RBP motifs (threshold 0.7). (b, c) ACO1 and SFRS13A were knocked down with specific siRNAs in MDA‐MB‐231 cells and exosomes respectively, and qRT‐PCR was used to evaluate miR‐148b‐3p expression level in cells and exosomes. (d) The miRNA pulldown assay of interactions between miR‐148b‐3p or mutated miR‐148b‐3p and ACO1 in the nucleus, cytoplasm and exosomes. Biotin‐poly (G) was used for negative control. (e) Anti‐ACO1 antibody and IgG as a negative control were used for the RIP assay. qRT‐PCR was used to detect miR‐148b‐3p levels in lysates from cells or exosomes of MDA‐MB‐231. The lysates from cells or exosomes were saved as input. The enrichment levels of miR‐148b‐3p in the coimmunoprecipitant complex are presented as percent input sample (% input). The Student's t‐test was adopted to analyze the statistical significance of the difference between two groups, and one‐way ANOVA test was utilized to analyze multiple groups (*p < 0.05, **p < 0.01).
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