Specificity of Four Monoclonal Anti-Nkx6-1 Antibodies

Klinck, Rasmus; Serup, Palle; Madsen, Ole; Jørgensen, Mette C.

doi:10.1369/jhc.7a7350.2008

Cited by 7 publications

(5 citation statements)

References 18 publications

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“…Islet histology procedures have been described [ 19 , 20 ]. Briefly, the antibodies used were as follows: insulin (Invitrogen; 18-0067; used at 1 : 800), glucagon (Cell Signaling Technologies; 8233; used at 1 : 400), Aldh1a3 (Novus Biologicals; NBP2-15339; used at 1 : 100), and Nkx6.1 (F55A12; 1 : 200; [ 21 ]). All antibodies were incubated overnight at 4°C and detected with either Alexa Fluor secondary conjugation (Alexa 488, 594), except for Nkx6.1, which was detected using a biotin/streptavidin exposure method.…”

Section: Methodsmentioning

confidence: 99%

db/dbMice Exhibit Features of Human Type 2 Diabetes That Are Not Present in Weight-Matched C57BL/6J Mice Fed a Western Diet

Burke

Batdorf

Burk

et al. 2017

Journal of Diabetes Research

114

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To understand features of human obesity and type 2 diabetes mellitus (T2D) that can be recapitulated in the mouse, we compared C57BL/6J mice fed a Western-style diet (WD) to weight-matched genetically obese leptin receptor-deficient mice (db/db). All mice were monitored for changes in body composition, glycemia, and total body mass. To objectively compare diet-induced and genetic models of obesity, tissue analyses were conducted using mice with similar body mass. We found that adipose tissue inflammation was present in both models of obesity. In addition, distinct alterations in metabolic flexibility were evident between WD-fed mice and db/db mice. Circulating insulin levels are elevated in each model of obesity, while glucagon was increased only in the db/db mice. Although both WD-fed and db/db mice exhibited adaptive increases in islet size, the db/db mice also displayed augmented islet expression of the dedifferentiation marker Aldh1a3 and reduced nuclear presence of the transcription factor Nkx6.1. Based on the collective results put forth herein, we conclude that db/db mice capture key features of human T2D that do not occur in WD-fed C57BL/6J mice of comparable body mass.

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Section: Methodsmentioning

confidence: 99%

db/dbMice Exhibit Features of Human Type 2 Diabetes That Are Not Present in Weight-Matched C57BL/6J Mice Fed a Western Diet

Burke

Batdorf

Burk

et al. 2017

Journal of Diabetes Research

114

View full text Add to dashboard Cite

show abstract

“…Primary antibodies were diluted in 0.1% TritronX-100 in PBS and applied to the cells, followed by incubation overnight at 4 °C. The following primary antibodies were used: goat polyclonal anti-Sox17 (1:1000) (R&D Systems, AF1924), mouse polyclonal anti-Oct3/4 (1:500) (Santa Cruz, sc5279), goat polyclonal anti-Pdx1 (1:8000) (Abcam, #ab47383), and mouse anti-Nkx6.1 (1:500) (Hybridoma bank, F55A10) (Klinck et al [ 46 ] ). Subsequently, cells were rinsed three times with PBS for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Enhanced Differentiation of Human Embryonic Stem Cells Toward Definitive Endoderm on Ultrahigh Aspect Ratio Nanopillars

Rasmussen

Reynolds

Petersen

et al. 2015

Adv Funct Materials

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Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have highlighted that the properties of the physical environment, such as substrate stiffness, affect cellular behavior. Here, mass‐produced, injection molded polycarbonate nanopillars are presented, where the surface mechanical properties, i.e., stiffness, can be controlled by the geometric design of the ultrahigh aspect ratio nanopillars (stiffness can be reduced by 25.0003). It is found that tall nanopillars, yielding softer surfaces, significantly enhance the induction of definitive endoderm cells from pluripotent human embryonic stem cells, resulting in more consistent differentiation of a pure population compared to planar control. By contrast, further differentiation toward the pancreatic endoderm is less successful on “soft” pillars when compared to “stiff” pillars or control, indicating differential cues during the different stages of differentiation. To accompany the mechanical properties of the nanopillars, the concept of surface shear modulus is introduced to describe the characteristics of engineered elastic surfaces through micro or nanopatterning. This provides a framework whereby comparisons can be drawn between such materials and bulk elastomeric materials.

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“…Antibodies used were from the following sources: iNOS (Santa Cruz Biotechnology), ␤-actin (Cell Signaling Technology), IB␣ (Santa Cruz Biotechnology), tubulin (Cell Signaling Technology), and Pdx-1 (Cell Signaling Technology). The antibody recognizing Nkx6.1 has been described (34). Immunoblot images were quantified using Image Lab software (Bio-Rad).…”

Section: Cell Culture Islet Isolation Adenoviral Vectors and Reagementioning

confidence: 99%

IL-1β reciprocally regulates chemokine and insulin secretion in pancreatic β-cells via NF-κB

Burke

Stadler

et al. 2015

American Journal of Physiology-Endocrinology and Metabolism

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Proinflammatory cytokines impact islet ␤-cell mass and function by altering the transcriptional activity within pancreatic ␤-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1␤, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-B controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1␤. Nitric oxide production, which is markedly elevated in pancreatic ␤-cells exposed to IL-1␤, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1␤-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1␤ were dependent on NF-B transcriptional activity. We conclude that IL-1␤-induced transcriptional reprogramming via NF-B reciprocally regulates chemokine and insulin secretion while also negatively regulating ␤-cell proliferation. These findings are consistent with NF-B as a major regulatory node controlling inflammation-associated alterations in islet ␤-cell function and mass. chemokine; inflammation; insulin secretion; interleukin-1; nitric oxide THE PROGRESSION TO BOTH TYPE 1 (T1DM) and type 2 diabetes mellitus (T2DM) proceeds via immune cell-associated alterations in islet ␤-cell mass and function. Alterations in islet ␤-cell mass and function are two major determinants controlling the total amount of insulin produced and secreted in response to physiological stimuli (e.g., glucose). Proinflammatory cytokines such as IL-1␤ and IFN␥ contribute significantly to losses in islet ␤-cell viability and insulin secretion. Islet

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Specificity of Four Monoclonal Anti-Nkx6-1 Antibodies

Cited by 7 publications

References 18 publications

db/dbMice Exhibit Features of Human Type 2 Diabetes That Are Not Present in Weight-Matched C57BL/6J Mice Fed a Western Diet

db/dbMice Exhibit Features of Human Type 2 Diabetes That Are Not Present in Weight-Matched C57BL/6J Mice Fed a Western Diet

Enhanced Differentiation of Human Embryonic Stem Cells Toward Definitive Endoderm on Ultrahigh Aspect Ratio Nanopillars

IL-1β reciprocally regulates chemokine and insulin secretion in pancreatic β-cells via NF-κB

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