An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes

Isidor, Marie S.; Winther, Sally; Basse, Astrid L.; Petersen, M. Christine H.; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B.

doi:10.1080/21623945.2015.1111972

Cited by 46 publications

(56 citation statements)

References 27 publications

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“…To study the possibly different roles of the two PKM isoforms, we performed knockdown experiments with siRNAs in mature WT‐1 brown adipocytes . The WT‐1 brown preadipocytes were differentiated into mature adipocytes, transfected with control siRNA (universal negative siRNA, siNEG), siPKM1, or siPKM2, and harvested four days later.…”

Section: Resultsmentioning

confidence: 99%

“…Primary brown adipocytes from interscapular, cervical, and axillary BAT from 3‐ to 4‐week‐old NMRI mice of either sex were isolated as described . The cell pellet was resuspended in culture medium and plated in 6‐well plates.…”

Section: Methodsmentioning

confidence: 99%

“…Mature adipocytes (day 6 for WT‐1 cells and day 8 for primary brown cultures) were reverse‐transfected with small interfering RNAs (siRNAs) as described . The final concentrations of transfection reagent and siRNA were 5 μL·mL −1 and 50 n m , respectively, for WT‐1 cells replated into 96‐well plates, and 9 μL·mL −1 and 90 n m , respectively, for primary brown adipocytes replated into 48‐well plates.…”

Section: Methodsmentioning

confidence: 99%

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Pyruvate kinase M2 represses thermogenic gene expression in brown adipocytes

et al. 2019

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Edited by L aszl o NagyUtilizing the thermogenic capacity of brown adipose tissue is a potential antiobesity strategy; therefore, the mechanisms controlling expression of thermogenesis-related genes are of interest. Pyruvate kinase (PK) catalyzes the last step of glycolysis and exists as four isoenzymes: PK, liver, PK, red blood cell, PK, muscle (PKM1 and PKM2). PKM2 has both glycolytic and nuclear functions. Here, we report that PKM2 is enriched in brown adipose compared with white adipose tissue. Specific knockdown of PKM2 in mature brown adipocytes demonstrates that silencing of PKM2 does not lead to a decrease in PK activity, but causes a robust upregulation of thermogenic uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) gene expression. This increase is not mediated by any of the known mechanisms for PKM2regulated gene expression, thus implying the existence of a novel mechanism for PKM2-dependent effects on gene expression.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Pyruvate kinase M2 represses thermogenic gene expression in brown adipocytes

et al. 2019

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“…(5) BATkl2 cells were amendable to siRNA transfection and showed highly efficient gene knockdown in the growth state as well as in differentiating adipocytes without the need of cell detachment (Isidor et al 2016). In addition, transfection of undifferentiated BATkl2 cells with plasmid DNA reached an efficiency which is probably sufficient for reporter assays.…”

Section: Discussionmentioning

confidence: 99%

A novel growth factor-dependent thermogenic brown adipocyte cell line from defined precursor cells

Kindler

Schweizer

et al. 2019

Preprint

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Molecular pathways regulating brown adipocyte formation and metabolism can be exploited as targets for the treatment of obesity and disorders of glucose and lipid metabolism such as type-2 diabetes. Investigations in this direction require adequate cell models for brown adipocytes and their precursors. We report the establishment of a novel clonal cell line derived from defined Lin -Sca1 + adipocyte precursors from murine interscapular brown fat. In contrast to most currently available lines, immortalization was achieved by serial passaging without viral or genetic manipulation. Instead, the media were supplemented with basic fibroblast growth factor, which was required for the maintenance of stable long-term growth and immature morphology. BATkl2 cells differentiated to adipocytes with high efficiency upon standard adipogenic induction independently of PPARg agonists and even at higher passage numbers. BATkl2 adipocytes showed readily detectable Uncoupling protein 1 (Ucp1) protein expression and acutely responded to norepinephrine with increased Ucp1 mRNA expression, lipolysis and uncoupled mitochondrial respiration. Highly efficient siRNAmediated knockdown was demonstrated in the growth state as well as in differentiating adipocytes, whereas plasmid DNA transfection was achieved in immature cells. These features make the BATkl2 cell line an attractive brown (pre)-adipocyte cell model.

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“…Adipocytes then were maintained in normal growth media for 24 hours before undergoing reverse transfection of control and Srsf1 siRNA 79 the differentiation and reverse transfection protocol was followed as described above, but adipocytes were seeded into 12 well plates.…”

Section: Cell Culture and Reverse Transfectionmentioning

confidence: 99%

Integrated analysis of a compendium of RNA-Seq datasets for splicing factors

Jin

Deng

et al. 2020

Preprint

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A vast amount of public RNA-sequencing datasets have been generated and used widely to study transcriptome mechanisms. These data offer precious opportunity for advancing biological research in transcriptome studies such as alternative splicing. We report the first large-scale integrated analysis of RNA-Seq data of splicing factors for systematically identifying key factors in diseases and biological processes. We analyzed 1,321 RNA-Seq libraries of various mouse tissues and cell lines, comprising more than 6.6 TB sequences from 75 independent studies that experimentally manipulated 56 splicing factors. Using these data, RNA splicing signatures and gene expression signatures were computed, and signature comparison analysis identified a list of key splicing factors in Rett syndrome and cold-induced thermogenesis. We show that coldinduced RNA-binding proteins rescue the neurite outgrowth defects in Rett syndrome using neuronal morphology analysis, and we also reveal that SRSF1 and PTBP1 are required for energy expenditure in adipocytes using metabolic flux analysis. Our study provides an integrated analysis for identifying key factors in diseases and biological processes and highlights the importance of public data resources for identifying hypotheses for experimental testing.

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An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes

Cited by 46 publications

References 27 publications

Pyruvate kinase M2 represses thermogenic gene expression in brown adipocytes

Pyruvate kinase M2 represses thermogenic gene expression in brown adipocytes

A novel growth factor-dependent thermogenic brown adipocyte cell line from defined precursor cells

Integrated analysis of a compendium of RNA-Seq datasets for splicing factors

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