MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors

Kato et al. 10.1073/pnas.0611192104.

Supporting Information

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Fig. 6. Amplification of genomic region of far upstream E-box enhancer of Col1a2 gene. (A) Schematic diagram of the far upstream region of Col1a2 gene. Three E-boxes are noted in the enhancer region located 16-kb upstream of the Col1a2 gene promoter transcription start site. This enhancer region was amplified by PCR using primers indicated by the arrows. (B) Amplified PCR fragment was cloned into pCR3.1 (Invitrogen) to confirm the sequence. Three E-box elements are identified (consensus CAxxTG, CACCT, or CAGGT). E-boxes are underlined.

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Fig. 7. Efficacy of U6 promoter-driven shRNA against dEF1. pCR3.1EGFP-dEF1shRNA was transfected into the mouse cell line TCMK-1. RNA and protein expression levels were quantified by real time qPCR (A) and Western blot analysis (B), respectively. A significant decrease of dEF1 mRNA levels was induced (by >90%) by shRNA against dEF1 but not control scrambled shRNA (#, P < 0.01) (A). Protein levels of dEF1 were also decreased by >70% (B) .

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Fig. 8. Alignment of hsa-miR-192 and mmu-miR-192 with human SIP1-3' UTR and mouse SIP1-3' UTR based on software from Memorial Sloan-Kettering Cancer Center (http://cbio.mskcc.org). The same target site also was identified by other software, MIRANDA and TargetScan, at Wellcome Trust Sanger Institute (http://microrna.sanger.ac.uk/index.shtml). Seven nucleotides in the 5' region of miR-192 (human and mouse) contain a perfect match with the 3' UTR sequence of the human and mouse SIP1 genes. Numbers next to the SIP1 3' UTR sequence are relative to the start sites of the 3' UTR. Sequences of these regions were 100% conserved in human, mouse, and rat. miR-215 (shown below in Fig. 8) also has a high complementarity to the same region and is conserved in human, mouse, and rat.

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Fig. 9. Dissociation curve of real-time qPCR of miR-194 (standards and mouse kidney samples). Only a single peak was observed.

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Fig. 10. Amplification of miR-194 (standards and mouse kidney samples).

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Fig. 11. miR-215 Mimic does not decrease SIP-1 3' UTR-S luciferase activity. The SIP1 3' UTR luciferase reporter (0.5 mg) was cotransfected with either the miR-215 Mimic (+ miR-215 Mimic) or the negative control mimic (NC Mimic) in MMC. No decrease in luciferase activity with miR-215 mimic was observed with the SIP1 3' UTR-S (sense) construct and no change with the control constructs (without SIP1 3' UTR, Control) or with SIP1 3' UTR antisense construct (AS).