Journal: Circulation. Heart failure

Article Title: Acute Targeting of General Transcription Factor IIB Restricts Cardiac Hypertrophy via Selective Inhibition of Gene Transcription

doi: 10.1161/CIRCHEARTFAILURE.114.001660

Figure Lengend Snippet: a. RNA was extracted from the neonatal, adult, and TAC hearts and subjected to qPCR for the indicated genes. The results were averaged and plotted as relative mRNA change (n=3). Error bars represent standard error of the mean (SEM) and * is p < 0.05 v. sham-operated or normal heart. b. Protein was extracted from the same heart tissue as in (a.) and analyzed by Western blots for the indicated antibodies. c. The Western blot signals for the different proteins were quantified, averaged, and plotted. Error bars represent standard error of the mean (SEM) and * is p < 0.05 v. sham-operated or normal adult (n=3, 2/3 samples shown in the blot). d. IGB images showing the RNA pol II density (y-axis) aligned across the Gtf2b (TFIIB) and Cdk9 genes (x-axis) in the neonatal, adult, and TAC hearts. Arrows positioned at the gene’s start site point to direction of transcription. e. Pol II (P) and H3K9ac (A) densities for the TFIIB and Cdk9 genes were plotted as the Log2 values of TAC/adult (T/A) and neonatal/adult (N/A) of the pol II in promoter proximal (PrP, −300 to +300), the in-gene (IP, +300 to end), and the downstream (DP, end to +5000), and of the H3K9ac in promoter proximal (PrA, −1000 to +1000), the in-gene (IA, +1000 to end) region, were plotted.

Article Snippet: TFIIB accession # {"type":"entrez-nucleotide","attrs":{"text":"NM_001514","term_id":"169259765","term_text":"NM_001514"}} NM_001514 (Origene) and CDK9 accession # {"type":"entrez-nucleotide","attrs":{"text":"NM_001261.2","term_id":"17017983","term_text":"NM_001261.2"}} NM_001261.2 (Origene) human cDNA with or without the 3’UTR containing the miR-1 target site were cloned into adenovirus.

Techniques: Western Blot

Journal: Circulation. Heart failure

Article Title: Acute Targeting of General Transcription Factor IIB Restricts Cardiac Hypertrophy via Selective Inhibition of Gene Transcription

doi: 10.1161/CIRCHEARTFAILURE.114.001660

Figure Lengend Snippet: a. TargetScan and Pictar miR-1 predicted targeting sites in the 3’UTR of Gtf2b and Cdk9. b. Cardiac myocytes were infected with the specified multiplicity of infection (moi) of adenoviral (Ad) vectors Ad.miR-1, Ad.miR-133, Ad.antimiR-1, or a control virus for 12 h before adding Ad.CMV.GFP.IRES.Luc. Twenty-four hours later, protein was extracted and analyzed by Western blots. c. Cardiac myocytes were simultaneously infected with the indicated Ad vectors. After the specified intervals, protein was extracted and analyzed by Western blots. d. The Western blot signals in (b.), and 2 other blots, for the different proteins, were quantified, averaged, and plotted (n=3). Error bars represent standard error of the mean (SEM) and * is p < 0.05 v. control. e. The Western blot signals in (c.), and 2 other blots, for the different proteins, were quantified, averaged, and plotted (n=3). Error bars represent standard error of the mean (SEM) and * is p < 0.05 v. control. f.–g. The full-length (FL) cDNA or mutants lacking the miR-1 target site (ΔmiR-1) of f. Cdk9 or g. TFIIB were designed, cloned, and equal doses expressed in growth-arrested cardiac myocytes. Protein was extracted, analyzed by Western blots, the signal quantified and normalized, and listed below the blots (n=2). h–i. LNA-modified antimiR-1 (15 mg/Kg), or a nonsense control, was injected into mice via the tail vein. h. Hearts were isolated after 7 days and analyzed by Western blots for the indicated proteins (n=3). i. The same set of hearts was analyzed by qPCR for the indicated miRNA and genes and the results plotted as fold change of antimiR-1/control hearts. Error bars represent standard error of the mean (SEM) and * is p < 0.05 v. control.

Article Snippet: TFIIB accession # {"type":"entrez-nucleotide","attrs":{"text":"NM_001514","term_id":"169259765","term_text":"NM_001514"}} NM_001514 (Origene) and CDK9 accession # {"type":"entrez-nucleotide","attrs":{"text":"NM_001261.2","term_id":"17017983","term_text":"NM_001261.2"}} NM_001261.2 (Origene) human cDNA with or without the 3’UTR containing the miR-1 target site were cloned into adenovirus.

Techniques: Infection, Control, Virus, Western Blot, Clone Assay, Modification, Injection, Isolation

Journal: Circulation. Heart failure

Article Title: Acute Targeting of General Transcription Factor IIB Restricts Cardiac Hypertrophy via Selective Inhibition of Gene Transcription

doi: 10.1161/CIRCHEARTFAILURE.114.001660

Figure Lengend Snippet: a. RNA extracted from neonatal myocytes supplemented with a control or Ad.miR-1 for 24 h was analyzed by qPCR. The data for 5 genes are averaged and plotted as relative miR-1/control values (n=3). Error bars represent standard error of the mean (SEM) and * is p < 0.05 v. control. b. Protein was extracted from cells treated as in (a.) in the absence or presence of exogenous Ad.TFIIB, or with a shRNA targeting TFIIB (Ad.shTFIIB) in the absence or presence of Ad.antimiR-1, and analyzed by Western blotting. c. RNA extracted from neonatal myocytes supplemented with a control or Ad.shTFIIB for 24 h was analyzed by qPCR (n=3). The data for 6 genes are averaged and plotted as relative shTFIIB/control values. Error bars represent standard error of the mean (SEM) and * is p < 0.05 v. control. d. RNA was extracted from cells treated as in (a.) in the absence or presence of exogenous Ad.TFIIB, and analyzed by qPCR for the specified genes (n=3). e. Myocytes were treated with control, Ad.miR-1, Ad.Cdk9, or Ad.miR-1+Cdk9 for 24. Cells were then fixed and subjected to ChIP-qPCR encompassing the TSS of the specified genes. The results were averaged and plotted as relative bound pol II. * is p < 0.05 v. control. f. Myocytes were treated with control (10 moi), Ad.miR-1 (5 or 10 moi, upper pannels), or Ad.shTFIIB (5 or 10 moi, lower panels) for 24 h before stimulating them with 100 nM endothelin (ET-1). After 24 h the cells were fixed and stained with Dapi and phalloidin.

Article Snippet: TFIIB accession # {"type":"entrez-nucleotide","attrs":{"text":"NM_001514","term_id":"169259765","term_text":"NM_001514"}} NM_001514 (Origene) and CDK9 accession # {"type":"entrez-nucleotide","attrs":{"text":"NM_001261.2","term_id":"17017983","term_text":"NM_001261.2"}} NM_001261.2 (Origene) human cDNA with or without the 3’UTR containing the miR-1 target site were cloned into adenovirus.

Techniques: Control, shRNA, Western Blot, ChIP-qPCR, Staining

Journal:

Article Title: Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks

doi: 10.1073/pnas.0510764103

Figure Lengend Snippet: Overexpression of TAF4b in GCs. (A) Immunoblotting of total cell extracts from stable f-TAF4b and flag control cell lines was performed to determine levels of TFIID subunits and general transcription factors by using anti-Pol II, anti-TAF1, anti-TAF4, anti-TFIIB, anti-Flag, and an anti-TAF4b monoclonal antibody. Anti-GAPDH antibodies were used to confirm equal loading. (B) Nuclear localization of Flag-TAF4b in SIGCs was detected by indirect immunofluorescence using an anti-Flag antibody. Nuclei were counterstained with Hoechst dye to label DNA. Arrows indicate nucleoli. (C) Partially purified nuclear extracts from f-TAF4b cells were precipitated with a control and anti-TAF4b polyclonal antibodies, followed by immunoblotting with anti-TAF4, anti-Flag, and anti-TBP antibodies.

Article Snippet: Western blot analysis was performed with anti-Pol II (H-224; Santa Cruz Biotechnology), anti-TAF4, anti-TFIIB (BD Transduction Laboratories), anti-Flag M2 (Sigma), anti-GAPDH (Abcam), anti-TAF1, and anti-TAF4b antibodies.

Techniques: Over Expression, Western Blot, Immunofluorescence, Purification

Journal:

Article Title: Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks

doi: 10.1073/pnas.0510764103

Figure Lengend Snippet: Phenotype of SIGCs and ovaries that lack TAF4b expression. (A) SIGCs were treated with TAF4b and control siRNAs for the indicated time points, followed by immunoblot analysis using anti-TAF4, anti-TAF4b, anti-c-Jun, and anti-β-tubulin antibodies as a loading control. (B) Treatment of SIGCs with TAF4b (+) in comparison with control (–) siRNAs reduces c-jun expression. Gene expression was determined by real-time RT-PCR analysis of RNAs from TAF4b siRNA-treated cells after standardization to control siRNAs and using Gapd as an internal control. Error bars represent SDs from three independent experiments. (C) Live-cell images of siRNA-treated SIGCs at ×10 magnification. An occasional fibroblastic cell can be identified in control siRNA-treated cells (arrows). (D) Hematoxylin-and-eosin-stained ovary sections are shown. Disorganized GCs surrounding an oocyte (Oo) are evident in Taf4b−/− follicles compared with wild-type Taf4b+/+ follicles, which are demarcated with a dotted line.

Article Snippet: Western blot analysis was performed with anti-Pol II (H-224; Santa Cruz Biotechnology), anti-TAF4, anti-TFIIB (BD Transduction Laboratories), anti-Flag M2 (Sigma), anti-GAPDH (Abcam), anti-TAF1, and anti-TAF4b antibodies.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Staining

Journal:

Article Title: Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks

doi: 10.1073/pnas.0510764103

Figure Lengend Snippet: Direct association of TFIID and c-Jun with target gene promoters. (A) Schematic representation of genomic loci analyzed by ChIP. At core promoter regions, only AP-1- and TFIID-binding sites are shown. For promoters (c-jun, Ccnd2, and Fst) that lack canonical TATA-box elements, TFIID-binding sites are depicted upstream of transcriptional start sites (not shown). For promoters (c-jun, Ccnd2, Fst, Inhba, and Vim) that have multiple predicted c-Jun/AP-1-binding sites, only one site is represented. Primer pairs (arrows) used to amplify a 5′ distal enhancer, core promoters, and a 3′ control region are shown. A box is used to represent each gene. (B) ChIP assays using stable f-TAF4b SIGC cells from genomic regions illustrated in A. Lane 1, control antibody; lane 2, anti-c-Jun; lane 3, anti-Flag; lane 4, anti-TAF4; lane 5, input. (C) ChIP assays from a stable f-TAF4b NIH/3T3 cell line. Lane 1, control antibody; lane 2, anti-c-Jun; lane 3; anti-Flag; lane 4, input. Inverted images of ethidium-bromide-stained PCR products are shown.

Article Snippet: Western blot analysis was performed with anti-Pol II (H-224; Santa Cruz Biotechnology), anti-TAF4, anti-TFIIB (BD Transduction Laboratories), anti-Flag M2 (Sigma), anti-GAPDH (Abcam), anti-TAF1, and anti-TAF4b antibodies.

Techniques: Binding Assay, Staining