13 research outputs found
The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein) Is Required for Preadipocyte Differentiation
<div><p>FTO (fat mass and obesity associated gene) was genetically identified to be associated with body mass index (BMI), presumably through functional regulation of energy homeostasis. However, the cellular and molecular mechanisms by which FTO functions remain largely unknown. Using 3T3-L1 preadipocyte as a model to study the role of FTO in adipogenesis, we demonstrated that FTO is functionally required for 3T3-L1 differentiation. FTO knock-down with siRNA inhibited preadipocyte differentiation, whereas ectopic over-expression of FTO enhanced the process. The demethylase activity of FTO is required for differentiation. Level of N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is decreased in cells over-expressing FTO. In contrast, overexpression of R96Q, a FTO missense mutant lack of demethylase activity, had no effect on cellular m<sup>6</sup>A level and impeded differentiation. Treatment with Rosiglitazone, a PPARγ agonist, could overcome the differentiation inhibition imposed by R96Q mutant, suggesting the effect of FTO is mediated through PPARγ.</p></div
Confirmation of microarray findings with RT-qPCR.
<p>(A) Three genes from the focal adhesion, cytoskeleton and ECM gene set (Lamc1, Dock1 and Thbs2) were selected and confirmed to be decreased 6hr following adipogenic induction in FTO knock-down cells. (B) Three PPARγ target genes (CD36, Fabp5 and Adiponectin) were selected and verified to be down-regulated 3 days following adipogenic induction in siFTO transfected cells. All gene expression data are represented as mean ± SD of triplicates. * stands for p<0.05, ** stands for p<0.01 and *** stands for p<0.001in Student’s t-test (siFTO versus siCtrl).</p
The demethylase activity of FTO is required during 3T3-L1 differentiation.
<p>Two lines of 3T3-L1 stably expressing wt FTO (hFTO-3 and hFTO-4) or R96Q (R96Q-2 and R96Q-4) were generated. (A) The protein expression level was confirmed by Western-blot using FTO and Myc antibodies, with β-actin as a loading control. (B) The sub-cellular localization of hFTO-4 or R96Q-4 was determined with confocal fluorescence imaging. Scale bars of 10μm are shown in the bottom row of images. (C) 3T3-L1differentiation was enhanced by over-expression of wt FTO and inhibited by R96Q. Level of differentiation was measured by extent of Oil Red-O staining at day 6 post differentiation induction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133788#sec002" target="_blank">materials and methods</a>). Demethylase activities of wt hFTO-4 and R96Q-4 in 3T3-L1 cells were determined by dot blotting (D) and quantified by Grayscale analysis with ImageJ software (E). Data are represented as means± SD of four replicates. * stands for p<0.05 and** stands for p<0.01in Student’s t-test.</p
Controlling of Ni-Based Composites in Salt Melt Synthesis with High Sodium-Ion Storage Performance
Owing to its fascinating properties (such as high theoretical
specific
capacity and considerable conductivity), nickel sulfide (NiS) was
investigated comprehensively as an anode material in sodium-ion batteries.
However, they still suffered from volume expansion and sluggish kinetics,
resulting in serious cycle capabilities. Herein, through controlling
the kind of molten salts (Na2SO4, NaCl, and
Na2CO3) in salt melt synthesis (SMS), a series
of NiS with an N, S-codoped carbon layer was successfully prepared,
accompanied with different morphologies and structures (earthworm-like
belts and triangular and spherical particles). Tailored by the ionic
strength and viscosity of molten salts, the as-prepared samples displayed
different crystallization behaviors, bringing about a difference in
electrochemical performance. As earthworm-like NiS@C was explored
as an anode material for SIBs, an initial capacity of 712.5 mAh g–1 at 0.5 A g–1 could be obtained,
and it still kept 527.4 mAh g–1 after 100 cycles.
Even at 2.0 A g–1, a capacity of 508.6 mAh g–1 could be achieved. Meanwhile, with the assistance
of detailed kinetic analysis, the rapid diffusion behaviors of Na+ and redox reaction mechanisms of as-fabricated samples were
proven for the enhanced electrochemical properties. Given this, this
work is expected to provide a method for designing the morphology
and structure of metal sulfides, while shedding light on the orientation
of fabricating advanced electrode materials for SIBs
Rosiglitazone rescued adipogenesis inhibition in 3T3-L1 cells expressing R96Q.
<p>(A) Oil Red-O staining of 3T3-L1 cell lines treated with MDI (top and middle row), or MDI plus Rosiglitazone (bottom row). Scale bars are 500μm in 40x images and 50μm in 400x images respectively (left column). (B) Level of differentiation in 3T3-L1 lines expressing wt hFTO-4 or R96Q-4, measured by the extent of Oil Red-O staining. Expression levels of five PPARγ target genes (aP-2, Fabp5, Adiponectin, Perilipin and CD36) were evaluated in 3T3-L1 over expressing wt hFTO-4 (C) or R96Q-4(D) at day 3 post differentiation induction. All gene expression data are represented as mean ± SD of triplicates.* stands for p<0.05, ** stands for p<0.01 and *** stands for p<0.001 in Student’s t-test (Rosig versus DMSO)</p
FTO knockdown inhibits differentiation of 3T3-L1 preadipocyte.
<p>(A) The mRNA levels of FTO, PPAR-γ, and β-actin at different time point during3T3-L1 differentiation was analyzed with RT-qPCR. The relative mRNA levels were determined by the ratio to that of PPAR-γ at time 0.The level of FTO mRNA (B) and protein (C) in cells treated with siRNAs and controls were assessed with RT-qPCR and Western Blot. (D) 3T3-L1 preadipocyte differentiation was inhibited by FTO knock-down. The extent of differentiation was assessed by Oil Red-O staining of intracellular triglyceride and measuring the extract’s absorbance at 540 nm. (E) Expression levels of PPAR-γ in 3T3-L1 cells and those transfected with control or FTO siRNAs at different time point during differentiation. (F) Expression levels of C/EBP-α in 3T3-L1 cells and those transfected with control or FTO siRNAs at different time point during differentiation. All gene expression data are represented as mean ± SD of triplicates.* stands for p<0.05 and ** stands for p<0.01 in Student’s t-test (siFTO vs. siCtrl).</p
Generation of the hormone-responsive human hepatoma cell line, AH-G6PC, and optimization of assay conditions for siRNA transfection.
<p>A. AH-G6PC cells express both the reporter gene, β-lactamase, under the control of the G6PC promoter and endogenous G6PC. B. Insulin dose-responsively decreases Dex/cAMP activation of β-lactamase activity. C. Changes in endogenous G6PC mRNA levels in AH-G6PC cells treated with vehicle (basal), Dex/cAMP, Dex/cAMP after transfection with control siRNAs (siControl), or Dex/cAMP after transfection with glucocorticoid receptor siRNAs (siGR), and increasing concentrations of insulin. D. Incubation of AH-G6PC cells with metformin (667 µM) for 16 h or rosiglitazone (10 uM) for 6 h reduces G6PC mRNA expression. Data are shown as the means ± SEM fold change relative to basal (no Dex/cAMP or insulin) in a study performed in triplicate, and is representative of multiple experiments. *, <i>P</i><0.05 by Student’s t-test vs. Dex/cAMP-treated samples.</p
Screening of a druggable siRNA library with a 4-gene High-Throughput-Genomics (HTG) assay.
<p>A. Layout of genes within each well of a 4-gene HTG 384-well ArrayPlate. B. Insulin dose-responsively inhibits Dex/cAMP induction of G6PC and PDK4 mRNA expression in AH-G6PC cells when assayed by the HTG platform. Data are presented as the means ± SEM of a study performed in triplicate; similar results were obtained in 4 independent experiments. C. Dual-flashlight plot comparing SSMD values vs. average fold change in G6PC mRNA expression by all siRNA pools in the library in order to select hits that regulate G6PC mRNA levels. Glucocorticoid receptor (GR) siRNAs were used as positive controls in each plates (red dots), while non-targeting siRNAs were used as negative controls in each plate (green dots). The blue square and the small rectangle represent the up-regulators with G6PC ≥2 and SSMD ≥1 and G6PC ≥1.3 and SSMD ≥2, respectively. The orange square and the small rectangle represent the down-regulators with G6PC≤1/2 and SSMD≤−1 and G6PC≤0.7 and SSMD≤−2, respectively. D. Major proximal molecular mediators and key negative modulators of hepatic insulin signaling. siRNA knockdown of target genes that lowered and increased G6PC mRNA expression are indicated in orange and blue, respectively.</p
Pharmacological inhibitor of glucocorticoid receptor (GR) led to similar reduction of gluconeogenesis gene expression to GR siRNA in AH-G6PC cell.
<p>AH-G6PC cells were preincubated with or without the GR antagonist RU486 (5 µM) for 18 h. The cells were then incubated with Dex/cAMP alone, Dex/cAMP plus 400 nM insulin, or Dex/cAMP plus RU486 (in the cells treated with RU486 overnight) for 6 h. A. Taqman analysis was performed on mRNA isolated from the cells to assay G6PC mRNA relative to Dex/cAMP treated cells. B. Multiplex HTG ArrayPlate analysis of mRNA isolated from the cells was used to quantify PEPCK, PGC1α, and G6PC mRNA expression. Data are plotted as fold change vs. mRNA levels in cells treated only with Dex/cAMP and are the means ± SEM of 3 independent experiments.</p
Schematic summary of the siRNA screening process that identified, confirmed, and characterized novel insulin sensitizer and GNG inhibitor target genes.
<p>Schematic summary of the siRNA screening process that identified, confirmed, and characterized novel insulin sensitizer and GNG inhibitor target genes.</p