Fig 1: miR-145 upregulation results in increased α-SMA expression and decreased KLF4 expression. (a) Transfection of miR-145 mimic, inhibitor, and control miRNA into VICs (n = 6/group) showed distinct transcriptomic profiles on hierarchical clustering (left) and separation between groups on principal component analysis (right). The miR-145 inhibitor transfected cells have similar profiles as control miRNA transfected cells, while miR-145 mimic transfected profile is distinct from the other two groups. (b) Volcano plot (left) showed that miR-145 mimic transfected cells resulted in an upregulation of α-SMA (ACTA2, red arrow pointing at the data point for ACTA2) compared to control sample (padj = 4 × 10−11) (middle). (c) Ingenuity Pathway Analysis Upstream Regulator Analysis (left) identified 11 upregulated genes (orange) and four that are downregulated genes (blue) that could regulate α-SMA expression after miR-145 mimic transfection. KLF4 is one of the genes with decreased normalized count after mimic transfection compared to control (padj = 0.011) (right).
Fig 2: miR-145 directly targets KLF4 to suppress KLF4 expression. (a) Luciferase assay confirms targeting of KLF4 by miR-145. Assay was performed using HEK cells with control miRNA (NC). n = 3 wild-type and 3 mutant vectors; one-way ANOVA, mean, and standard deviation shown. (b) Immunoblot confirmed that VIC co-culture with KLF4 protein decreases α-SMA protein expression (n = 7/group; paired t-test, mean, and standard deviation shown), and (c) KLF4 siRNA treatment resulted in an increase in α-SMA protein expression. C: Control (vehicle only), NC: Negative control siRNA, siRNA: KLF4 siRNA (n = 3/group; Kruskal–Wallis test, median, and interquartile shown). *: p < 0.05.
Fig 3: Schematic illustration depicting the mechanism of AST-pretreated MSCs treat AKI-CKD. By binding with the transcription factor KLF4 in MSCs, astilbin promotes the expression of PTGS2/COX2, enhances the transformation of AA to PGE2, promotes the M2-type polarisation of macrophages, and ultimately increases the efficacy of MSCs on AKI-CKD mice (This picture is created in BioRender)
Fig 4: KLF4, the target for AST, directly binds the PTGS2 promoter to promote its expression in MSCs. (A) Chemical structure of AST. (B) the DARTS assay detecting a marked increase in the 40–55 kD band following MSCs incubation in pronase-digested MSCs lysates, and (C) the molecular docking models of KLF4 and astilbin. (D) Real binding of KLF4 with astilbin confirmed by SPR assay. (E) Binding motif of KLF4 identified by WebLogo in the JASPAR online tools. (F) The PTGS2 promoter region to which KLF4 binds. (G) The ChIP-PCR results for PTGS2. (H) Dual-luciferase reporter assay results. The data are presented as the mean standard deviation, with ***p < 0.001
Supplier Page from Abcam for Recombinant human KLF4 protein