Name: an adipocyte cell culture model to study the impact of protein and micro-rna modulation on adipocyte function
Uploaded: 2021-05-04T14:00:00
Description: Watch this Scientific Journal Video about An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function at JoVE.com

This work was supported by INSERM, the Universit&#233; C&#244;te d&#39;Azur, and the French National Research Agency (ANR) through the program Investments for the future Laboratory of Excellence (Labex SIGNALIFE-ANR-11-LABX-0028-01) and Initiative of Excellence (Idex UCAJEDI ANR-15-IDEX-0001). J.J. is supported by grants from the Soci&#233;t&#233; Francophone du Diab&#232;te (SFD), the Association Fran&#231;aise d&#39;Etude et de Recherche sur l&#39;Ob&#233;sit&#233; (AFERO), the Institut Th&#233;matique Multi-Organismes Technologies pour la Sant&#233; (ITMO), and the Fondation Benjamin-Delessert. J.G. is supported by ANR-18-CE14-0035-01. J-F.T. is supported by ANR grant ADIPOPIEZO-19-CE14-0029-01 and a grant from the Fondation pour la Recherche M&#233;dicale (Equipe FRM, DEQ20180839587). We also thank the Imaging Core Facility of C3M funded by the Conseil D&#233;partemental des Alpes-Maritimes and the R&#233;gion PACA, which is also supported by the GIS IBiSA Microscopy and Imaging Platform C&#244;te d&#39;Azur (MICA).

NOTE: Use sterile techniques to perform all the steps of the protocol in a laminar flow cell culture hood. See Table of Materials for details about all reagents and equipment.
1. Differentiation of murine 3T3-L1 fibroblasts into adipocytes
<ol>
	<li>Grow the 3T3-L1 fibroblasts in 100 mm dishes in culture medium-DMEM without pyruvate, 25 mM glucose, 10% newborn calf serum, and 1% penicillin and streptomycin (Figure 1A). Place the dishes in a tiss...

<ol>
	<li>Kl&#246;ting, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-sensitive+obesity.">Insulin-sensitive obesity.</a> American Journal of Physiology, Endocrinology and Metabolism. 299 (3), 506-515 (2010).</li><li>Weyer, C., Foley, J. E., Bogardus, C., Tataranni, P. A., Pratley, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlarged+subcutaneous+abdominal+adipocyte+size,+but+not+obesity+itself,+predicts+type+II+diabetes+independent+of+insulin+resistance.">Enlarged subcutaneous abdominal adipocyte size, but not obesity itself, predicts type II diabetes independent of insulin resistance.</a> Diabetologia. 43 (12), 1498-1506 (2000).</li><li>Bl&#252;her, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+dysfunction+contributes+to+obesity+related+metabolic+diseases.">Adipose tissue dysfunction contributes to obesity related metabolic diseases.</a> Best Practice &amp; Research Clinical Endocrinology &amp; Metabolism. 27 (2), 163-177 (2013).</li><li>Lorente-Cebri&#225;n, S., Gonz&#225;lez-Muniesa, P., Milagro, F. I., Mart&#237;nez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+other+non-coding+RNAs+in+adipose+tissue+and+obesity:+emerging+roles+as+biomarkers+and+therapeutic+targets.">MicroRNAs and other non-coding RNAs in adipose tissue and obesity: emerging roles as biomarkers and therapeutic targets.</a> Clinical Science. 133 (1), 23-40 (2019).</li><li>Jager, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tpl2+kinase+is+upregulated+in+adipose+tissue+in+obesity+and+may+mediate+interleukin-1beta+and+tumor+necrosis+factor-{alpha}+effects+on+extracellular+signal-regulated+kinase+activation+and+lipolysis.">Tpl2 kinase is upregulated in adipose tissue in obesity and may mediate interleukin-1beta and tumor necrosis factor-{alpha} effects on extracellular signal-regulated kinase activation and lipolysis.</a> Diabetes. 59 (1), 61-70 (2010).</li><li>Vergoni, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+and+the+activation+of+the+p53+pathway+mediate+alterations+in+metabolic+and+secretory+functions+of+adipocytes.">DNA damage and the activation of the p53 pathway mediate alterations in metabolic and secretory functions of adipocytes.</a> Diabetes. 65 (10), 3062-3074 (2016).</li><li>Berthou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tpl2+kinase+regulates+the+COX-2/prostaglandin+E2+axis+in+adipocytes+in+inflammatory+conditions.">The Tpl2 kinase regulates the COX-2/prostaglandin E2 axis in adipocytes in inflammatory conditions.</a> Molecular Endocrinology. 29 (7), 1025-1036 (2015).</li><li>Ceppo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+the+Tpl2+kinase+in+inflammatory+changes+and+insulin+resistance+induced+by+the+interaction+between+adipocytes+and+macrophages.">Implication of the Tpl2 kinase in inflammatory changes and insulin resistance induced by the interaction between adipocytes and macrophages.</a> Endocrinology. 155 (3), 951-964 (2014).</li><li>Jager, J., Gr&#233;meaux, T., Cormont, M., Le Marchand-Brustel, Y., Tanti, J. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta-induced+insulin+resistance+in+adipocytes+through+down-regulation+of+insulin+receptor+substrate-1+expression.">Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression.</a> Endocrinology. 148 (1), 241-251 (2007).</li><li>Jager, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tpl2+kinase+is+upregulated+in+adipose+tissue+in+obesity+and+may+mediate+interleukin-1beta+and+tumor+necrosis+factor-{alpha}+effects+on+extracellular+signal-regulated+kinase+activation+and+lipolysis.">Tpl2 kinase is upregulated in adipose tissue in obesity and may mediate interleukin-1beta and tumor necrosis factor-{alpha} effects on extracellular signal-regulated kinase activation and lipolysis.</a> Diabetes. 59 (1), 61-70 (2010).</li><li>Hart, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-34a+as+hub+of+T+cell+regulation+networks.">miR-34a as hub of T cell regulation networks.</a> Journal of ImmunoTherapy of Cancer. 7, 187 (2019).</li><li>Brandenburger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-34a+is+differentially+expressed+in+dorsal+root+ganglia+in+a+rat+model+of+chronic+neuropathic+pain.">MiR-34a is differentially expressed in dorsal root ganglia in a rat model of chronic neuropathic pain.</a> Neuroscience Letters. 708, 134365 (2019).</li></ol>

This paper presents a detailed protocol for the differentiation and transfection of mature adipocytes. This reverse-transfection method is a simple, economical, and highly efficient method to transfect oligonucleotides such as, but not limited to, siRNAs, micro-RNA mimics, and anti-micro-RNAs into 3T3-L1 adipocytes, which is one of the most difficult cell lines to transfect. This method has some limitations that need to be considered. This protocol is not efficient for transfection with plasmid DNA, which limits the util...

Obesity is considered a major risk factor for numerous metabolic diseases, including insulin resistance (IR), Type 2 Diabetes (T2D), and cardiovascular diseases1. Current therapies have failed to stop the constantly rising prevalence of these diseases, and the management of the IR of obese and diabetic patients remains an important clinical issue. Adipose tissue plays a crucial role in the control of energy homeostasis, and its pathological expansion during obesity contributes to the development of IR and T2D2,3. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Many research studies have investigated the role of protein-coding RNAs in adipocyte physiology and their association with obesity.
More recently, the discovery of non-coding RNAs (ncRNAs), especially micro-RNAs (miRs), has forged novel concepts related to the mechanism of the regulation of gene expression programs. Studies have shown that ncRNAs are important regulators of adipocyte function, and that their dysregulation plays an important role in metabolic diseases4. Thus, the manipulation of proteins and ncRNAs in adipocytes is crucial to decipher their roles in adipocyte function and their impact on pathologies such as T2D. However, manipulating the expression of proteins and ncRNAs in vivo as well as in primary adipocytes remains highly challenging, favoring the use of in vitro adipocyte models.
Murine 3T3-L1 fibroblasts easily differentiate into mature, functional, and insulin-responsive adipocytes, which are a well-characterized cell line used to study adipocyte function (e.g., insulin signaling, glucose uptake, lipolysis and adipokines secretion)5,6,7,8,9,10. These properties make 3T3-L1 adipocytes an attractive model to modulate the expression of protein-coding and nc-RNAs to decipher their role in adipocyte function and their potential role in obesity-related diseases. Unfortunately, whereas 3T3-L1 fibroblasts are easy to transfect using commercially available reagents, differentiated 3T3-L1 adipocytes are one of the most difficult cell lines to transfect. This is why numerous studies manipulating gene expression in 3T3-L1 cells have focused on adipocyte differentiation rather than on adipocyte function.
For a long time, the only efficient technique to transfect adipocytes was electroporation5, which is tedious, expensive, and can cause cell damage. This paper reports a reverse-transfection technique using a common transfection reagent, which reduces hands-on time for transfection, has no effect on cell viability, and is much less expensive than electroporation. This protocol is perfectly suited for the transfection of siRNA and other oligonucleotides such as micro-RNA mimics (miR mimics) and anti-miRs. The principle of the reverse-transfection protocol is to incubate the transfection reagent and the oligonucleotides to form a complex in the cell culture plate and then seed the mature adipocytes into the wells. Then, the adipocytes reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. This simple, efficient, and inexpensive methodology permits the study of the role of protein-coding RNAs and miRs in adipocyte function and their potential role in obesity-related diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 well Tissue Culture Plate</td>
			<td>Dutscher</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsin (10x)</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>diluted to 5x with D-PBS</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Sigma</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Accell Non-targeting Pool</td>
			<td>Horizon Discovery</td>
			<td>D-001910-10-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type I from calf skin</td>
			<td>Sigma-Aldrich</td>
			<td>C8919</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s&#160; Modified Eagles&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>41965062</td>
			<td>4.5 g/L D-Glucose; L-Glutamine; no Pyruvate</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>51976</td>
			<td></td>
		</tr>
		<tr>
			<td>FAM-labeled Negative Control si-RNA</td>
			<td>Invitrogen</td>
			<td>AM4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Free Glycerol Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>F6428</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Standard Solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7793</td>
			<td></td>
		</tr>
		<tr>
			<td>HSP90 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-131119</td>
			<td>Dilution : 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Improved Minimal Essential Medium (Opti-MEM)</td>
			<td>Gibco</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Human Recombinant</td>
			<td>Gibco</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr>
			<td>miRIDIAN micro-RNA mimics</td>
			<td>Horizon Discovery</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript II RT Kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript Primer Assays Hs_RNU6-2_11</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript Primer Assays Mm_miR-34a_1</td>
			<td>Qiagen</td>
			<td>MS00001428</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR Kit</td>
			<td>Qiagen</td>
			<td>219073</td>
			<td></td>
		</tr>
		<tr>
			<td>Newborn Calf Serum</td>
			<td>Gibco</td>
			<td>16010-159</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr>
			<td>ON-TARGETplus Mouse Plin1 si-RNA SMARTpool</td>
			<td>Horizon Discovery</td>
			<td>L-056623-01-0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin and Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Perilipin-1 antibody</td>
			<td>Cell Signaling</td>
			<td>3470</td>
			<td>Dilution : 1/1000</td>
		</tr>
		<tr>
			<td>Petri dish 100 mm x 20 mm</td>
			<td>Dutscher</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>PKB antibody</td>
			<td>Cell Signaling</td>
			<td>9272</td>
			<td>Dilution : 1/1000</td>
		</tr>
		<tr>
			<td>PKB Phospho Thr308 antibody</td>
			<td>Cell Signaling</td>
			<td>9275</td>
			<td>Dilution : 1/1000</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent (INTERFERin)</td>
			<td>Polyplus</td>
			<td>409-10</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin antibody</td>
			<td>Sigma aldrich</td>
			<td>T6199</td>
			<td>Dilution : 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Vamp2 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB5136</td>
			<td>Dilution : 0.1 &#181;g/mL</td>
		</tr>
	</tbody>
</table>

an adipocyte cell culture model to study the impact of protein and micro-rna modulation on adipocyte function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Using the procedure of reverse-transfection described here to modulate the expression of proteins or micro-RNAs in 3T3-L1 adipocytes, the adipocytes have been shown to preserve their morphology after the transfection (Figure 1B,C). Indeed, 2 days after the transfection, the adipocytes were well-spread and attached to the plate and presented multilocular lipid droplets that are a characteristic of mature 3T3-L1 adipocytes. The lipid content wa...

Watch this Scientific Journal Video about An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function at JoVE.com

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

This protocol makes it possible to manipulate proteins, or microRNA expression, in differentiated adipocytes to study their role in adipocytes function. This reverse-transfection method is a simple, economical, and highly efficient method to transfect oligonucleotides into mouse 3T3-L1 adipocytes. This method can also be applied to human preadipocytes differentiated into adipocytes.Demonstrating the procedure will be Melanie Gaudfrin, a technician from our laboratory. Grow the 3T3-L1 fibroblasts in 100 millimeter dishes in DMEM without pyruvate, 25 millimolar glucose, 10%newborn calf serum, and one percent penicillin and streptomycin. Place the dishes in a tissue culture incubator at 37 degrees Celsius and seven percent carbon dioxide.Two days after confluence, change the culture medium, replacing it with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin supplemented with 0.25 millimolar IBMX, 0.25 micromolar dexamethasone, five micrograms per milliliter insulin, and 10 micromolar rosiglitazone. Incubate the dishes for two days. Two days later, replace the culture medium with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin supplemented with five micrograms per milliliter insulin and 10 micromolar rosiglitazone and incubate for another two days.Feed the cells every two days with DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin and keep the cells in the incubator. One day or a few hours before the transfection, prepare a solution of collagen type I at 100 micrograms per milliliter in 30%ethanol from a stock solution at one milligram per milliliter. Add 250 microliters of collagen per well of a 12-well plate or 125 microliters per well of a 24-well plate and spread the solution over the surface of the well.Leave the plate without the lid under the culture hood until the collagen dries. Then wash it twice with D-PBS. Pipette the siRNA with improved minimum essential medium to mix and incubate for five minutes at room temperature.Add the transfection reagent and the improved minimal essential medium to the siRNA and pipette to mix. Incubate it for 20 minutes at room temperature. Then add the transfection mix to each well of the collagen-coated plate.Wash the cells in the 100 millimeter Petri dish twice with D-PBS. Add 5x trypsin to the cells, making sure to cover the entire surface with the trypsin. Wait for 30 seconds and carefully remove the trypsin.Incubate the Petri dish for five to 10 minutes at 37 degrees Celsius in the incubator. Then tap the dish to detach the cells, avoiding cell damage. Add 10 milliliters of DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin to neutralize the trypsin.Carefully pipette the medium up and down to detach the cells and homogenize the cell suspension. Count the cells using a Malassez counting chamber or an automated cell counter and adjust the concentration of the cells to 0.625 million cells per milliliter of medium. Seed 800 microliters of the cell suspension per well of a 12-well plate or 400 microliters of the cell suspension per well of a 24-well plate containing the transfection mix.Incubate the plates in a cell culture incubator and do not disturb them for 24 hours. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 millimolar glucose, 10%FCS, and one percent penicillin and streptomycin and return the cells to the incubator. Typical time points for detecting target knockdown after siRNA or miR delivery are 24 to 48 hours for mRNA and 48 to 96 hours for protein.Functional experiments can be performed on transfected adipocytes including insulin signaling, glucose uptake, adipokine secretion, lipolysis, and lipogenesis. The adipocytes have been shown to preserve their morphology after the transfection. Two days after transfection, the adipocytes presented multilocular lipid droplets with lipid content not different between the transfected and non-transfected adipocytes.The mRNA expression of various differentiation markers was unchanged in transfected cells compared to that in non-transfected adipocytes. The reverse-transfection protocol is efficient as more than 70%of the adipocytes were transfected. Three days after the transfection with siRNA against Perilipin-1, the mRNA level of Perilipin-1 had decreased by 70%and the protein level by 63%Perilipin-1 expression was also analyzed by fluorescence microscopy four days after the transfection and was found to have decreased by 92%compared to its expression in control adipocytes.The reverse-transfection of adipocytes with microRNA mimicking oligonucleotides to up-regulate the expression of miR-34a is shown here. The over-expression of miR-34a led to the decrease in VAMP2 protein expression by 50%The knockdown of Perilipin-1 in 3T3-L1 adipocytes led to an increase in basal lipolysis while the over-expression of miR-34a led to the inhibition of insulin-induced protein kinase B phosphorylation and glucose uptake. It's important to reach a high level of adipocytes differentiation to perform the transfection on newly differentiated adipocytes and to carefully monitor the treatment of adipocytes with trypsin.Following the transfection, it is possible to perform functional experiments on the adipocytes to study the impact of proteins or microRNA manipulation on insulin signaling, glucose uptake, lipogenesis, and lipolysis.

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Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...