Journal: Endocrinology

Article Title: Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation

doi: 10.1210/en.2016-1183

Figure Lengend Snippet: Genes Involved in Unfolded Protein Response/ER Stress and Transcription Factor Complex Are Elevated in the Pituitaries of Cga −/− Mice

Article Snippet: Cloning of Tceal5 in situ hybridization (ISH) probe, Tceal5-EGFP transgene We amplified a 336-bp piece of the Tceal5 cDNA (ENSMUST00000066819; primers, 5-TCTCTTCCAGGTACCAGCTACCAGC-3 and 5-TCTAGCTTGCCCTGGCGTGC-3) from a 8-week-old Cga −/− pituitary cDNA and TA-cloned it into the pGEM-T-Easy vector (Promega).

Techniques: Amplification, Sequencing, Scaffolding, Variant Assay, Binding Assay

Journal: Endocrinology

Article Title: Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation

doi: 10.1210/en.2016-1183

Figure Lengend Snippet: Characterization of TCEAL5 in the pituitary and in a progenitor cell. A, Tceal5 expression is increased from birth to adulthood in the Cga−/− pituitary. Absolute quantification with qPCR showed an incremental increase in Tceal5 expression from birth through 4 and 8 weeks of age in 50-ng cDNA; P ≤ .011 for all pairs. B, Tceal5 transcripts are localized in the anterior and intermediate lobes of the pituitary. Tceal5 is expressed in few AL and IL cells in the wild-type, and Cga−/− pituitaries show an altered distribution within these lobes. Purple stain marks Tceal5, whereas green is nuclear counterstaining. Scale bar, 50 μm in all panels. C, Cga−/− but not wild-type pituitaries have Tceal5-positive thyrotropes. Combined Tceal5 ISH (purple) with TSHB IHC (brown) and green nuclear counterstaining. In a typical view, yellow arrows mark some Tceal5+/TSHB−, and black arrows mark Tceal5+/TSHB+ cells. Note that single Tceal5+ cells are typically close to thyrotrope clusters. D, A small portion of proliferating cells express Tceal5. Proliferation detected with EdU (green) and Tceal5 mRNA (purple) over nuclear staining with DAPI (blue). Representative views from both genotypes showing cells with arrows as EdU+/Tceal5+ (black), Edu+/Tceal5− (yellow) and EdU−/Tceal5+ (red), respectively. E, Transgenic TCEAL5-EGFP has nuclear expression in the Pit1-triple pituitary progenitor cell line. Individual cells were imaged with confocal microscopy to determine the localization of EGFP in stable, transgenic cell populations. EGFP without TCEAL5 was localized to the cytoplasm, whereas the TCEAL5-GFP fusion localized to the nucleus. Scale bar, 10 μm. F, Stable overexpression of Tceal5 significantly enhances proliferation in a pituitary progenitor cell line. The Pit1-triple line expresses Pou1f1, Gh, Prl, Tshb, and Cga (38). We created heterologous lines stably expressing a Tceal5-Egfp gene fusion or Egfp only and plated the same number of cells on day 0. Six plates with each were monitored and 3 were harvested for cell counting after 4 or 8 days. G, Stable overexpression of Tceal5 does not change the apoptosis rate in a pituitary progenitor line. Nine wells in a cluster plate were assayed for apoptosis with the TUNEL assay heterologous cell lines stably expressing Tceal5-Egfp gene fusion or Egfp only. Nuclei were counterstained with DAPI, and the percentage of TUNEL+ cells was compared. NS, not significant.

Article Snippet: Cloning of Tceal5 in situ hybridization (ISH) probe, Tceal5-EGFP transgene We amplified a 336-bp piece of the Tceal5 cDNA (ENSMUST00000066819; primers, 5-TCTCTTCCAGGTACCAGCTACCAGC-3 and 5-TCTAGCTTGCCCTGGCGTGC-3) from a 8-week-old Cga −/− pituitary cDNA and TA-cloned it into the pGEM-T-Easy vector (Promega).

Techniques: Expressing, Staining, Transgenic Assay, Confocal Microscopy, Over Expression, Stable Transfection, Cell Counting, TUNEL Assay

Journal: Endocrinology

Article Title: Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation

doi: 10.1210/en.2016-1183

Figure Lengend Snippet: Protein sequence comparison suggests similar and divergent functions for the mouse TCEAL5. A, Conserved domain comparison for mouse TCEAL proteins (NCBI Conserved Domain search, http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). TCEAL proteins share the BEX domain and TCEAL3/6 include an approximately 70-amino acid-long segment similar to the conserved DUF2042 domain, which has an unknown function. B, The mouse TCEAL5 protein is highly conserved among mammals. Q5H9L2, Q8CCT4, E2RDN6, H2R4M4, and F6UAE4 protein sequences from the UniProt database (http://www.uniprot.org/) for human, mouse, dog, chimpanzee, and horse, respectively. Symbols mark the following: asterisk, identical; colon/semicolon, similar; hyphen, missing amino acids. The BEX domain is marked with magenta box. C, Functional domain comparison suggests different functions in proliferation for TCEAL5 compared with related proteins. Alignment was created with K-Align from the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/msa/kalign/) with default settings and coloring of individual amino acid residues followed the Clustal X color scheme (http://www.jalview.org/help/html/colourSchemes/clustal.html). Magenta, translucent blue, and green boxes are BEX domain, proapoptotic, and regulatory functional domain, respectively.

Article Snippet: Cloning of Tceal5 in situ hybridization (ISH) probe, Tceal5-EGFP transgene We amplified a 336-bp piece of the Tceal5 cDNA (ENSMUST00000066819; primers, 5-TCTCTTCCAGGTACCAGCTACCAGC-3 and 5-TCTAGCTTGCCCTGGCGTGC-3) from a 8-week-old Cga −/− pituitary cDNA and TA-cloned it into the pGEM-T-Easy vector (Promega).

Techniques: Sequencing, Functional Assay