Ethacrynic acid activates the IL-17/MAPK pathways during the induction of AML cell differentiation. (A) Flow cytometry analysis of CD14 and CD11b expression of U937 cells treated with ATRA (0.2 μM), ethacrynic acid, and khellin (30 μM) for 3 days. (B) Quantitative analysis of results in (A). (C,D) Flow cytometry analysis of CD1b or CD14 expression of U937 cells treated with ATRA (0.2 μM), ethacrynic acid, and khellin (30 μM) for 3 days. (E) Volcano plot of the differential gene expression between DMSO- and ethacrynic-acid-treated groups from three biological replicates. (F) Numbers of up- and downregulated genes in the ethacrynic acid group relative to the DMSO group (greater than twofold change, adj. p ≤ 0.05). (G) GO enrichment analysis of differentially expressed genes. (H) KEGG enrichment analysis of differentially expressed genes. (I–M) GSEA of the expression profile of U937 cells treated with DMSO and ethacrynic acid using various signaling signatures. (N) Heatmap of DEGs involved in the IL-17 signaling pathway between the DMSO and ethacrynic acid groups. (O) RT-qPCR analysis showing mRNA expression of IL-17 B and D and IL-17 RA, RB, and RC in the ethacrynic acid group compared to the DMSO group. (P) Heatmap of DEGs involved in the IL-17/MAPK signaling pathways between the DMSO and ethacrynic acid groups. (Q) RT-qPCR analysis showing mRNA expression of FOSB, FOLS1, JUNB, JUND, MMP1, S1009A, HSPB1, HSPA8, CCL2, and MMP9 in the ethacrynic acid group compared to the DMSO group. Results in (B,O,Q) are represented as mean ± SEM, n = 3, t-test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001. DEG, differential gene expression.
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