Journal: Biosensors & bioelectronics

Article Title: Bioelectronic sensor mimicking the human neuroendocrine system for the detection of hypothalamic-pituitary-adrenal axis hormones in human blood.

doi: 10.1016/j.bios.2020.112071

Figure Lengend Snippet: Fig. 1. Schematic of the HPA axis of the human neuroendocrine system and bio electronic sensor system. In the human HPA axis, the hypothalamus, pituitary, and adre nal cortex communicate with interactions between CRHR1 or MC2R and CRH or ACTH, respectively. The bioelectronic sensor system mimics this system, in which CRHR1- and MC2R-embedded nanodiscs (CRHR1NDs and MC2RNDs) are immobilized on the floating electrode-based CNT-FET, and applied to detect CRH and ACTH, respectively.

Article Snippet: CRHR1 and MC2R cDNA were purchased from Origene (USA), and the MSP1E3D1 bacterial expression vector (pMSP1E3D1) was purchased from Addgene (USA).

Techniques:

Journal: Biosensors & bioelectronics

Article Title: Bioelectronic sensor mimicking the human neuroendocrine system for the detection of hypothalamic-pituitary-adrenal axis hormones in human blood.

doi: 10.1016/j.bios.2020.112071

Figure Lengend Snippet: Fig. 2. Characterization of CRHR1 and MC2R expressed in HEK-293 cells and E. coli (a) Dose- dependent responses of CRHR1 expressed in HEK- 293 cells to CRH (n ¼ 3, *p < 0.05, **p < 0.01, ***p < 0.001). (b) Selectivity of CRHR1 expressed in HEK- 293 cells after the addition of various hormones and neurotransmitters (n ¼ 3, CRH: corticotropin- releasing hormone, ACTH: adrenocorticotropic hor mone, DA: dopamine, EP: epinephrine, SE: seroto nin). (c) Dose-dependent responses of MC2R expressed in HEK-293 cells to ACTH (n ¼ 3, *p < 0.05, **p < 0.01, ***p < 0.001). (d) Selectivity of MC2R expressed in HEK-293 cells to various hor mones and neurotransmitters (n ¼ 3). (e) Western blot analysis of CRHR1 and MC2R expressed in E. coli using V5 Ab. The band indicates the molecular weight of CRHR1 (left image). The band corresponds to the molecular weight of MC2R (right image). (f) FE-SEM image of CRHR1NDs. The NDs had a dis coidal shape, and their average size was approxi mately 20 nm.

Article Snippet: CRHR1 and MC2R cDNA were purchased from Origene (USA), and the MSP1E3D1 bacterial expression vector (pMSP1E3D1) was purchased from Addgene (USA).

Techniques: Western Blot, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: DHCR24, a Key Enzyme of Cholesterol Synthesis, Serves as a Marker Gene of the Mouse Adrenal Gland Inner Cortex

doi: 10.3390/ijms24020933

Figure Lengend Snippet: Phenotypic analyses of Dhcr24 cKO mice. ( A ) Oil Red O staining showing lipid droplets in the adrenal glands of euthyroid P35 mice. ( B ) Plasma levels of corticosterone, ACTH, and the ACTH/corticosterone ratio in euthyroid P35 mice. Each dot (WT) and circle (cKO) represents data from one mouse. Data are shown with the mean. Scale bars, 210 µm. WT: wild-type mice, cKO: Dhcr24 conditional knockout mice. ns, no significant difference.

Article Snippet: Hormones were measured using the corticosterone EIA kit (K014-H1, Arbor Assays, Ann Arbor, MI, USA) and the mouse ACTH ELISA kit (NBP3-14759, Novus Biologicals, Centennial, CO, USA) according to manufacturers’ instructions.

Techniques: Staining, Clinical Proteomics, Knock-Out

Journal: Endocrine-Related Cancer

Article Title: Effects of epigenetic pathway inhibitors on corticotroph tumour AtT20 cells

doi: 10.1530/ERC-19-0448

Figure Lengend Snippet: POMC expression and ACTH secretion after JQ1 treatment. QRT-PCR analysis confirmed the significant down-regulation of Pomc transcription after 48 h JQ1 treatment (A). Down-regulation of POMC protein expression was also confirmed by Western blot analyses at 24 h, 48 h and 72 h after JQ1 treatment, compared to JQ1- treated cells; POMC is observed as two bands corresponding to pre-pro-POMC and pro-POMC (calnexin was used as a loading control) (B). Densitometry analysis confirmed that POMC expression was significantly down-regulated at 24 h, 48 h and 72 h (C). Concentrations of ACTH in the media from the same cells, which reflected ACTH secretion by these cells, significantly decreased at 48 h and 72 h after treatment with JQ1, when compared to JQ1- treated cells (D). DMSO or JQ1-treated cells were used as negative controls, with qRT-PCR experiments performed in n = 4 technical and n = 4 biological replicates, and Western blot and ELISAs performed in n = 4 biological replicates. Statistical significance is relative to JQ1- treatment with * P < 0.05, ** P < 0.005 and *** P < 0.0005.

Article Snippet: Briefly, media was diluted 1:3 in distilled water and 200 μL added per well of a mouse/rat ACTH ELISA kit (Origene, MD, USA), incubated for 4 h with biotinylated and enzyme-labelled antibodies, before visualising with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate and reading at 405 nM on a Pherastar FS (BMG LabTech, Ortenberg, Germany).

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

Journal: Antioxidants & redox signaling

Article Title: Pterostilbene Decreases the Antioxidant Defenses of Aggressive Cancer Cells In Vivo: A Physiological Glucocorticoids- and Nrf2-Dependent Mechanism.

doi: 10.1089/ars.2015.6437

Figure Lengend Snippet: FIG. 3. Pter bioavailability in brain and the pituitary gland and its effect on ACTH synthesis and POMC ex- pression in AtT-20 cells. (A) Whole brain and pituitary gland levels of Pter after its i.v. administration (30 mg/kg) (n = 6–7 mice; *Significantly different p < 0.01, comparing hypophysis vs. whole brain levels). Data in (A) correspond to tissues obtained from nontumor-bearing mice (results obtained in A2058 or MeWo melanoma-bearing mice were not significantly different, not shown). (B) Effect of Pter on ACTH production by AtT-20 cells in a 24 h-period (mea- sured between 24 and 48 h or between 48 and 72 h). (C) Effect of Pter on POMC expression and (D) POMC protein levels (72 h of culture time) in AtT-20 cells (n = 6 for B, C, and D; *Significantly different p < 0.01, comparing +Pter vs. controls). For the in vitro experiments (B, C, and D), Pter (15 lM, see under the Results section) and CRH (100 nM) were added at time 0 and every 24 h through the culture time and were present, after each addition, for only 30 min. After the 30-min period, culture flasks were washed out (thrice with PBS) and the medium was renewed (controls received identical treatment). POMC, proopiomelanocortin.

Article Snippet: For Western blotting (see also above), POMC mouse monoclonal antibody (clone 2B2) from Origen was used.

Techniques: Expressing, In Vitro

Journal: Oncotarget

Article Title: Analysis of pituitary adenoma expression patterns suggests a potential role for the NeuroD1 transcription factor in neuroendocrine tumor-targeting therapies

doi: 10.18632/oncotarget.26513

Figure Lengend Snippet: Average hormone and NeuroD1 expression values

Article Snippet: For immunohistochemical staining, confocal microscopy, and electron immunocytochemistry, the following primary antibodies were used: mouse monoclonal ACTH antibody, diluted 1:500 (clone AH26, Diagnostic BioSystems, Netherlands) rabbit polyclonal TSH antibody, RTU (Cell Marque, USA) mouse monoclonal FSH antibody, diluted 1:100 (clone С10, DAKO, Denmark) mouse monoclonal LH antibody, diluted 1:500 (clone С93, DAKO, Denmark) rabbitpolyclonal GH antibody, diluted 1:100 (BioGenex, USA) rabbit polyclonal PRL antibody, diluted 1:700 (DAKO, Denmark) mouse monoclonal NeuroD1 antibody, diluted 1:1000 (clone ab60704, Abcam, United Kingdom) mouse monoclonal Ki-67antibody, diluted 1:200 (clone MIB-1, DAKOCytomation, Denmark) mouse monoclonal CK7antibody, diluted 1:300 (clone OV-TL 12/30, DAKO, Denmark) The following secondary antibodies/reagents were used for immunohistochemical staining: mouse EnVisionTM+ System, Peroxidase (DAKO, Denmark) rabbit EnVisionTM+ System, Peroxidase (DAKO, Denmark) MultiVision Polymer Cocktail (Thermo Scientific, UK) The following secondary antibodies were used for confocal microscopy: Alexa Fluor 647 goat anti-Mouse, diluted 1:100 (Abcam, UK) Alexa Fluor 488 goat anti-Rabbit, diluted 1:100 (Abcam, UK) The following secondary antibodies were used for electron immunocytochemistry: goat-anti mouse antibody conjugated to 10nm colloidal gold, diluted 1:100 (Sigma-Aldrich, US) goat-anti rabbit antibody conjugated to 5nm colloidal gold, diluted 1:100 (Sigma-Aldrich, US)

Techniques: Expressing

Journal: Oncotarget

Article Title: Analysis of pituitary adenoma expression patterns suggests a potential role for the NeuroD1 transcription factor in neuroendocrine tumor-targeting therapies

doi: 10.18632/oncotarget.26513

Figure Lengend Snippet: Percentage of antigen expressing cells in normal adenohypophysis fragments taken near adenoma boundaries

Article Snippet: For immunohistochemical staining, confocal microscopy, and electron immunocytochemistry, the following primary antibodies were used: mouse monoclonal ACTH antibody, diluted 1:500 (clone AH26, Diagnostic BioSystems, Netherlands) rabbit polyclonal TSH antibody, RTU (Cell Marque, USA) mouse monoclonal FSH antibody, diluted 1:100 (clone С10, DAKO, Denmark) mouse monoclonal LH antibody, diluted 1:500 (clone С93, DAKO, Denmark) rabbitpolyclonal GH antibody, diluted 1:100 (BioGenex, USA) rabbit polyclonal PRL antibody, diluted 1:700 (DAKO, Denmark) mouse monoclonal NeuroD1 antibody, diluted 1:1000 (clone ab60704, Abcam, United Kingdom) mouse monoclonal Ki-67antibody, diluted 1:200 (clone MIB-1, DAKOCytomation, Denmark) mouse monoclonal CK7antibody, diluted 1:300 (clone OV-TL 12/30, DAKO, Denmark) The following secondary antibodies/reagents were used for immunohistochemical staining: mouse EnVisionTM+ System, Peroxidase (DAKO, Denmark) rabbit EnVisionTM+ System, Peroxidase (DAKO, Denmark) MultiVision Polymer Cocktail (Thermo Scientific, UK) The following secondary antibodies were used for confocal microscopy: Alexa Fluor 647 goat anti-Mouse, diluted 1:100 (Abcam, UK) Alexa Fluor 488 goat anti-Rabbit, diluted 1:100 (Abcam, UK) The following secondary antibodies were used for electron immunocytochemistry: goat-anti mouse antibody conjugated to 10nm colloidal gold, diluted 1:100 (Sigma-Aldrich, US) goat-anti rabbit antibody conjugated to 5nm colloidal gold, diluted 1:100 (Sigma-Aldrich, US)

Techniques: Expressing

Journal: Oncotarget

Article Title: Analysis of pituitary adenoma expression patterns suggests a potential role for the NeuroD1 transcription factor in neuroendocrine tumor-targeting therapies

doi: 10.18632/oncotarget.26513

Figure Lengend Snippet: Percentage of antigen expressing cells in normal adenohypophysis

Article Snippet: For immunohistochemical staining, confocal microscopy, and electron immunocytochemistry, the following primary antibodies were used: mouse monoclonal ACTH antibody, diluted 1:500 (clone AH26, Diagnostic BioSystems, Netherlands) rabbit polyclonal TSH antibody, RTU (Cell Marque, USA) mouse monoclonal FSH antibody, diluted 1:100 (clone С10, DAKO, Denmark) mouse monoclonal LH antibody, diluted 1:500 (clone С93, DAKO, Denmark) rabbitpolyclonal GH antibody, diluted 1:100 (BioGenex, USA) rabbit polyclonal PRL antibody, diluted 1:700 (DAKO, Denmark) mouse monoclonal NeuroD1 antibody, diluted 1:1000 (clone ab60704, Abcam, United Kingdom) mouse monoclonal Ki-67antibody, diluted 1:200 (clone MIB-1, DAKOCytomation, Denmark) mouse monoclonal CK7antibody, diluted 1:300 (clone OV-TL 12/30, DAKO, Denmark) The following secondary antibodies/reagents were used for immunohistochemical staining: mouse EnVisionTM+ System, Peroxidase (DAKO, Denmark) rabbit EnVisionTM+ System, Peroxidase (DAKO, Denmark) MultiVision Polymer Cocktail (Thermo Scientific, UK) The following secondary antibodies were used for confocal microscopy: Alexa Fluor 647 goat anti-Mouse, diluted 1:100 (Abcam, UK) Alexa Fluor 488 goat anti-Rabbit, diluted 1:100 (Abcam, UK) The following secondary antibodies were used for electron immunocytochemistry: goat-anti mouse antibody conjugated to 10nm colloidal gold, diluted 1:100 (Sigma-Aldrich, US) goat-anti rabbit antibody conjugated to 5nm colloidal gold, diluted 1:100 (Sigma-Aldrich, US)

Techniques: Expressing