Fig 1: Analysis of VDR-RXR repressor complex. (A) The structural construct of the VDR-RXR repressor complex. The complex shows VDR (cyan) and RXR (marine blue) heterodimer. VDA (red) bound with VDR and RXR heterodimer interacts with miR193a promoter and recruits SMRT (pink) and HDAC3 (magenta) and forms a repressor complex. (B) A schematic diagram is displaying the formation of the VDR-RXR repressor complex at the miR193a promoter. The VDA bound VDR makes a heterodimer with RXR and recruits SMRT and HDAC3 and forms a repressor complex. (C) Protein blots of three different cellular lysates of UPDs and DPDs were probed for RXR, VDR, SMRT, HDAC3, and GAPDH. Gels of three independent lysates are displayed. (D–G). Cumulative densitometric data (protein GAPDH) are shown as dot plots. * p < 0.05 compared with respective UPD. (H) Cellular lysates from protocol C were immunoprecipitated (IP) with the RXR antibody. Protein blots of RXR-IP fractions were probed for RXR, VDR, SMRT, HDAC3, and IgG. Gels are displayed. (I–L). Cumulative densitometric data (protein/GAPDH) from the protocol H, are shown as dot plots. * p < 0.05 compared with respective UPD.
Fig 2: Determination of repressors contributing to the downing of miR193a expression. (A) Vitamin D receptor- Retinoid X receptor (VDR-RXR) heterodimer-miR193a complex. DNA binding domain (DBD) of RXR (Marine blue) and substrate-binding domain (SBD) of RXR (Marine blue) are involved in binding with DNA (miR193a). Some interacting residues of RXR-DBD and RXR-SBD are represented in Magenta color, and VDR-DBD and VDR-SBD are represented in Brown color. VDR substrate-binding domain (SBD) and DNA binding domain (DBD) is represented in Cyan color. (B) DPDs were transfected with either siRNA-VDR or siRNA-RXR (n = 3). After 48 h, RNAs were extracted from control (C) and siRNA-VDR- and siRNA-RXR-transfected DPDs. RNAs were assayed for miR193a. Cumulative data from three independent experiments are shown in bar graphs. * p < 0.05 compared with control (C). (C). WT1 recruits EZH2 and binds at miR193a promoter. WT1 (Marine blue) binds on miR193a promoter with interacting residues (Magenta). (D). DPDs were treated with buffer or transfected with siRNA-WT1 (n = 3). After 48 h, RNAs were extracted from control (C) and siRNA-WT1-transfected DPDs. RNAs were assayed for miR193a. Cumulative data from three independent experiments are shown in a bar diagram. * p < 0.05 compared with control (C).
Fig 3: Transcription factor binding on miR193a promoter. (A). DR1, DR3 and DR4 elements for nuclear receptor binding. (B). Sox2 motif on the miR193a gene.
Fig 4: Evaluation of the effect of VDA on downing of miR193a and associated expression of podocyte molecular markers. (A) UPDs (at 33 °C) and DPDs (at 37 °C) were incubated in media containing either buffer (Control, C) or VDA (EB1089, 100 nM) for 48 h (n = 3). RNAs were assayed for miR193a expression. Cumulative data of three independent experiments are displayed in a bar diagram. * p < 0.05 compared with respective control (C). (B) B. cDNAs were prepared from RNA extracted from the protocol A, amplified with specific primers for WT1, VDR, APOL1, CD2AP, and Nephrin. Cumulative data are shown as bar graphs. * p < 0.05 compared respective controls (C).
Fig 5: APOL1 3′ UTR and miR193a Secondary structures. (A) miR193a-5p structure conformation and hybrid structure conformations of APOL1 3′ UTR and miR193a-5p. The APOL1 3’ UTR structure conformation consists of secondary structure elements such as External loop, Helices, bulge loop, Interior loop, and Hairpin loops. i. The hybrid structure is showing miR193a-5p and APOL1 3′ UTR pairing with a 3′ overhang. ii. The hybrid structure conformation shows intensive pairing. (B) Tertiary structure of APOL1 3′ UTR and miR193a-5p (Magenta) complex. The miR193a-5p binding site on APOL1 3′ UTR is represented in Cyan. (C) UPDs were transfected with either control plasmid (empty vector, EV) or a specific inhibitor of miR193a (25 nM) (n = 3). In parallel sets of experiments, DPDs were transfected with either control plasmid (EV) or miR193a plasmid (50 nm) (n = 3). RNAs were assayed for miR193a. Cumulative data are displayed in bar graphs. * p < 0.0 compared to respective EV. (D) cDNAs from EV- and miR193a inhibitor-treated UPDs from 7C were amplified with specific primers for APOL1, VDR, WT1, and Nephrin (n = 3). Cumulative data are shown in a bar diagram. * p < 0.05 compared to respective EV. (E) cDNAs from EV and miR193a-transfected DPDs from 7C were amplified with specific primers for APOL1, VDR, WT1, and Nephrin (n = 3). Cumulative data are shown in bar graphs. * p < 0.05 compared with respective EV.
Supplier Page from OriGene Technologies for MIR193a Human MicroRNA Expression Plasmid (MI0000487)