Journal: Nature medicine

Article Title: Thioredoxin-interacting protein regulates insulin transcription through microRNA-204.

doi: 10.1038/nm.3287

Figure Lengend Snippet: Figure 3 MAFA as a target of miR-204. (a,b) The effects of miR-204 on Mafa mRNA (a) and protein (b) expression as assessed by qRT-PCR and immunoblotting. (c) Changes in the Mafa occupancy of the insulin promoter in response to miR-204 as measured by ChIP. IgG control results are shown on the right. Gapdh, glyceraldehyde 3-phosphate dehydrogenase. (d) Alignment of the miR-204 seed sequence (arrow) and the wild-type (WT) rat Mafa 3′ UTR target sequence (bold) and the mutated (M) target sequence (gray). (e) miR-204–directed repression of the luciferase reporter gene bearing the WT or mutant Mafa 3′ UTR segments as assessed 24 h after cotransfecting INS-1 cells with the wild-type or mutant reporter plasmids. All data are shown as the mean ± s.e.m. of three independent experiments, and one representative immunoblot is shown. *P < 0.05, **P < 0.01 (Student’s t test). NS, not significant.

Article Snippet: Five micrograms of rabbit antibody to MAFA (A300611A, Bethyl Laboratories, Montgomery, TX) or normal rabbit IgG (sc2027, Santa Cruz) were used for immuno precipitation, and purified DNA fragments were quantified by qPCR with the primers listed in Supplementary Table 2.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Sequencing, Luciferase, Mutagenesis

Journal: Nature medicine

Article Title: Thioredoxin-interacting protein regulates insulin transcription through microRNA-204.

doi: 10.1038/nm.3287

Figure Lengend Snippet: Figure 4 The effects of TXNIP on MAFA and insulin production. (a–c) TXNIP-overexpressing INS-TXNIP cells analyzed for Mafa mRNA by qRT-PCR (a), Mafa protein by immunoblotting (b) and Mafa occupancy of the insulin promoter by ChIP (c) compared to INS-LacZ control cells. IgG control is shown on the right. (d) MAFA mRNA expression, assessed by qRT-PCR, in human islets that were transfected with a TXNIP expression plasmid or LacZ. (e,f) Insulin mRNA, as assessed by qRT-PCR, in INS-TXNIP cells (e) and human islets transfected with TXNIP (f). (g,h) Insulin content (g) and insulin secretion (h) assessed by ELISA in INS-TXNIP and INS-LacZ control cells. (i) Islet insulin content measured by ELISA in primary islets of TXNIP-deficient HcB-19 and control C3H mice. (j) Insulin content, measured by ELISA and normalized for DNA content, in control C3H and TXNIP-deficient HcB-19 mouse islets that were transfected with scrambled control microRNA or precursor miR-204 and analyzed 2 d later. All data are shown as the mean ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 (Student’s t test (a–h) or analysis of variance (j)). NS, not significant.

Article Snippet: Five micrograms of rabbit antibody to MAFA (A300611A, Bethyl Laboratories, Montgomery, TX) or normal rabbit IgG (sc2027, Santa Cruz) were used for immuno precipitation, and purified DNA fragments were quantified by qPCR with the primers listed in Supplementary Table 2.

Techniques: Quantitative RT-PCR, Western Blot, Control, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay