Journal: EMBO Molecular Medicine

Article Title: Adipogenesis and insulin sensitivity in obesity are regulated by retinoid-related orphan receptor gamma

doi: 10.1002/emmm.201100172

Figure Lengend Snippet: Functional siRNA screen of potential RORγ target genes identified by microarray analysis of 3T3-L1 cells overexpressing RORγ. 3T3-L1 cells were transfected with siRNA pools, differentiated and fluorescently stained to assess the percentage of differentiated cells ( n = 4). Nuclear run-on assay for transcriptional activity of genes in undifferentiated 3T3-L1 cells infected with control or RORγ-overexpressing lentivirus. 32P-labelled mRNA was hybridized to cDNA from the indicated genes on a slot blot and analysed by autoradiography. Expression of Mmp3 mRNA in 3T3-L1 preadipocytes overexpressing RORγ by transient transfection or viral infection. MMP3 promoter constructs with potential ROR response elements (RREs) cloned into a luciferase reporter vector. Luciferase assay in 3T3-L1 preadipocytes overexpressing RORγ or control vector 48 h after transfection normalized to renilla luciferase activity. Activation of IL17 promoter was used as a control ( n = 4). Chromatin immunoprecipitation of RORγ in 3T3-L1 cells using anti-HA antibody 6 days after infection with RORγ or control lentivirus. DNA was quantified by qPCR using specific primers for RRE1 and RRE2 and normalized to input ( n = 3). Fluorescent staining of differentiated 3T3-L1 adipocytes expressing RORγ or control vector during differentiation treated with or without 20 µM of MMP3 inhibitor 2 days before and after induction of differentiation (scale: 100 µm). The results shown are representative of three independent experiments. Values represent means ± SD, * p ≤ 0.05 and ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: mRNA was isolated and transcribed into cDNA using the Multi-MACS cDNA kit (Miltenyi). mRNA expression was assessed by real-time PCR using SybrGreen (Invitrogen) according to the manufacturer's protocol. mRNA expression was normalized to 36b4 or Gapdh.

Techniques: Functional Assay, Microarray, Transfection, Staining, Nuclear Run-on Assay, Activity Assay, Infection, Control, Dot Blot, Autoradiography, Expressing, Construct, Clone Assay, Luciferase, Plasmid Preparation, Activation Assay, Chromatin Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of platelet-derived growth factor in the vascular walls of patients with lower extremity arterial occlusive disease

doi: 10.3892/etm.2015.2275

Figure Lengend Snippet: Reagents used in the study.

Article Snippet: Multi-functional DNA Gel Extraction kit II (Spin-Column) , BioTeke Corporation, Beijing, China.

Techniques: RNA Extraction, Gel Extraction

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: The chromatin-binding protein Smyd1 restricts adult mammalian heart growth

doi: 10.1152/ajpheart.00235.2016

Figure Lengend Snippet: Transcriptome analyses reveal Smyd1 to be a transcriptional repressor of a core set of developmental genes. Transcriptome analyses were performed separately on right ventricles (RV) and left ventricles (LV) from mice with deletion of Smyd1 at 2 wk and 9 wk after removal from tamoxifen diet (controls were normal diet-fed littermates). A: all genes measured in both groups, RV and LV, are displayed in heat map format (red is upregulation, green downregulation, and black statistically unchanged) and clustered according to similar behavior. 2 clusters were defined as indicated, and the functionality of genes in those clusters was determined by gene ontology (GO) analysis. The networks for cluster 1 (B) and 2 (C) for the biological process ontology are shown. In the network figures, node size indicates the P value, and linkage between two terms indicates the relatedness, as determined by κ statistics. The color indicates functional groups with each group represented by their most significant leading term. All terms shown in the network image of cluster 1 are filtered at P < 0.005; those in cluster 2 are filtered at P < 0.0001. Bioinformatic analyses of transcripts with altered expression in the LV, at week 2 (D) and week 9 (E) after Tmx treatment, show significant enrichment in genes involved in extracellular matrix remodeling, fibrosis, and transcriptional repression. MF, molecular function; BP = biological process; CC, cellular component; KEGG, KEGG analysis; ITP, Interpro analysis. Microarray results for several of these transcripts were subsequently validated by RT-PCR (F: all those we attempted to validate are shown; n = 3–6/group; *P < 0.05 vs no tamoxifen group). Chromatin immunoprecipitation (ChIP) and qPCR for Smyd1 (using FLAG antibody) show enrichment in the promoter region of target genes tgfbeta3 (G) and nppa (H); however, no corresponding enrichment of histone H3 lysine K4 trimethylation was detected in these regions (bottom). *P ≤ 0.05. I: luciferase reporter assay using the tgfbeta3 and nppa promoters confirms that Smyd1 acts as a transcriptional repressor by inhibiting transcription of these genes; n = 6/group; *P < 0.05. J: as a negative control, Smyd1 is enriched by ChIP-PCR at neither β-tubulin nor β-actin using primers shown previously to target the regulatory regions upstream of these genes [−3 kb for β-tubulin (24), −73 bp for β-actin (29)].

Article Snippet: After 24 h, the cells were transfected with 75 ng of human TGF-β3, Nppa, negative control (scrambled), or positive control (actin) luciferase reporter construct (SwitchGear Genomics) using FuGene HD (SwitchGear Genomics) at a 6:1 ratio to DNA and incubated for 48 h. Luciferase activity was assayed using the LightSwitch Luciferase assay reagent (SwitchGear Genomics) according to the manufacturer's instructions and measured using a BioTek Synergy Neo HTS Multi-Mode microplate reader.

Techniques: Functional Assay, Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Negative Control