Summary
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.
Abstract
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.
Introduction
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.
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Protocol
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
- 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 tissue culture incubator (7% CO2 and 37 °C).
- Two days after confluence, change the culture medium, replacing with DMEM without pyruvate, 25 mM glucose, 10% fetal calf serum (FCS), and 1% penicillin and streptomycin supplemented with 0.25 mM 3-Isobutyl-1-methylxanthine (IBMX), 0.25 µM dexamethasone, 5 µg/mL insulin, and 10 µM rosiglitazone.
NOTE: It takes 5 days to reach confluency when the cells are seeded at 300,000 cells per 100 mm dish. - Two days later, replace the culture medium with DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin supplemented with 5 µg/mL insulin and 10 µM rosiglitazone and incubate for 2 days. Then, feed the cells every 2 days with DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin (Figure 1B).
- Transfect the 3T3-L1 adipocytes 7-8 days after the beginning of the differentiation protocol.
NOTE: It is important to reach a high level of differentiation (>80%) before the transfection to avoid the proliferation of the remaining fibroblasts after the transfection, which would lead to a mixed population of cells that might bias the results.
2. Preparation of precoated plates
- On the day before or a few hours before the transfection, prepare a solution of collagen type I at 100 µg/mL in 30% ethanol from a stock solution at 1 mg/mL. Add 250 µL of collagen per well of a 12-well plate and 125 µL 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. Wash twice with Dulbecco's phosphate-buffered saline (D-PBS).
NOTE: Precoated plates are available for purchase.
3. Preparation of the transfection mix
NOTE: The final concentration of siRNA is between 1 and 100 nM (1 to 100 pmol of siRNA per well of a 12-well plate). The final concentration of the miR mimic is 10 nM (10 pmol/well). Determine the best concentration of each siRNA, miR mimic, or other oligonucleotide prior to starting the experiment to avoid off-target effects. Perform transfection experiments in triplicate to facilitate statistical analysis of the results. Prepare all reagents in excess to account for normal loss during pipetting.
- Mix by pipetting (volume/volume) the siRNA (or other oligonucleotides) with improved Minimal Essential Medium (Table 1). Incubate for 5 min at room temperature.
- Add the transfection reagent and the improved Minimal Essential Medium to the siRNA, and pipet to mix (Table 1). Incubate for 20 min at room temperature (during this time, proceed to section 4). Add the transfection mix to each well of the collagen-coated plate.
4. Preparation of the 3T3-L1 adipocytes
- Wash the cells in the 100 mm Petri dish twice with D-PBS. Add 5x trypsin to the cells (1 mL per 100 mm dish), making sure to cover all of the surface with the trypsin. Wait for 30 s and carefully remove the trypsin.
- Incubate the Petri dish for 5-10 min at 37 °C in the incubator. Tap the 100 mm dish to detach the cells.
- Add 10 ml of DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin to neutralize the trypsin. Carefully pipet 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 6.25 x 105 cells/mL of medium. Seed 800 µL of the cell suspension/well of a 12-well plate (5 x 105 cells) or 400 µl of the cell suspension/well of a 24-well plate (2.5 x 105 cells) containing the transfection mix.
NOTE: One 100 mm Petri dish of adipocytes will allow the preparation of one 12-well plate or one 24-well plate. A 100 mm dish usually contains 6-7 x 106 adipocytes, which correspond to 5 x 105 adipocytes per well of a 12-well plate. - Incubate the plates in a cell culture incubator (7% CO2 and 37 °C), and do not disturb the cells for 24 h. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin.
NOTE: It is also possible to seed the cells into collagen-precoated 48- and 96-well plates but take more precautions when replacing the media to avoid detachment of the adipocytes.
5. Functional analysis of transfected 3T3-L1 adipocytes
- Study target knockdown 24-48 h and 48-96 h after siRNA or miR mimic delivery for mRNA and protein, respectively.
- Perform functional analyses of transfected adipocytes to study insulin signaling, glucose uptake, adipokine secretion, lipolysis, and lipogenesis.
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Representative Results
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 was not different between the transfected and non-transfected adipocytes (Figure 1D,E). Moreover, the mRNA expression of differentiation markers such as peroxisome proliferator-activated receptor gamma 2 (Pparγ2), adiponectin (Adipoq), glucose transporter 4 or solute carrier family 2 member 4 (Slc2a4), insulin receptor substrate 1 (Irs1), perilipin-1 (Plin1) was unchanged in transfected cells compared to that in non-transfected adipocytes (Figure 1F). This reverse-transfection protocol is efficient as >70% of the adipocytes were transfected (Figure 1G,H).
Perilipin-1 is an adipocyte-specific protein known to promote lipid droplet formation and inhibit lipolysis. Here, 3T3-L1 adipocytes were transfected with scrambled siRNA (si-SCR) or siRNA against Plin1 (si-PLIN1). Three days after the transfection with si-PLIN1, the mRNA level of Plin1 had decreased by 70% (Figure 2A) and the protein level by 63% (Figure 2B,C). PLIN1 expression was also analyzed by fluorescence microscopy 4 days after the transfection and was found to have decreased by 92% compared to its expression in control adipocytes (Figure 2D-F), thus demonstrating the efficacy of both the transfection protocol and the si-PLIN1.
This protocol was also used to perform reverse-transfection of adipocytes with micro-RNA mimicking (miR mimics) oligonucleotides to upregulate the expression of miR-34a (Figure 3A). The overexpression of miR-34a led to the decrease in VAMP2 protein expression by 50% (Figure 3B,C), a confirmed target of miR-34a11,12. Finally, this study shows that reverse-transfection of 3T3-L1 adipocytes preserves their function and responsiveness to insulin stimulation. Indeed, knockdown of Plin1 in 3T3-L1 adipocytes led to an increase in basal lipolysis (Figure 4A). Moreover, the overexpression of miR-34a in 3T3-L1 adipocytes led to the inhibition of insulin-induced protein kinase B phosphorylation (Figure 4B,C) and glucose uptake (Figure 4D).
Figure 1: Differentiation of 3T3-L1 fibroblasts into mature adipocytes. (A) 3T3-L1 fibroblasts were seeded at a density of 3 x 105 cells per 100 mm dish. Representative 10x brightfield image of 3T3-L1 fibroblasts 2 days later. (B) Two days after confluency (day 0), the 3T3-L1 fibroblasts were differentiated into adipocytes using a differentiation cocktail mix for 4 days (until day 4). Representative 10x brightfield image of 3T3-L1 fibroblasts differentiated into adipocytes (day 7). Adipocytes with multilocular lipid droplets are easily discernable. (C) The 3T3-L1 adipocytes were transfected with si-SCR on day 7. Representative 10x brightfield images of transfected 3T3-L1 adipocytes (day 9). The morphology of the transfected adipocytes is comparable to that of the non-transfected adipocytes, implying that the transfection method is gentle and not toxic to the adipocytes. Scale bars: 50 µm. (D–E) The 3T3-L1 adipocytes were transfected with si-SCR on day 7 (upper panels); non-transfected adipocytes (lower panels). Two days later, the cells were incubated with Oil Red O to stain lipids. (D) Representative images of the stained cells in the plate and representative 10x brightfield images are shown. Scale bars: 50 µm. (E) The Oil Red O incorporated into the cells was eluted with 2-propanol and quantified using a spectrophotometer. Data are expressed in arbitrary units, with the absorbance of the non-transfected cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed by Student’s t-test. (F) The 3T3-L1 adipocytes were transfected with si-SCR. Three days after the transfection, the cells were harvested to isolate total RNA. The expression of adipocyte differentiation markers was measured by qRT-PCR and normalized using 36B4 RNA levels. The data represent the mRNA expression in transfected cells relative to that in non-transfected cells (normalized to 1, represented by the dotted line) and are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed by Student’s t-test. (G–H) The 3T3-L1 adipocytes were transfected with si-SCR or fluorescent dye (FAM)-labeled si-RNA and plated on coverslips. 3T3-L1 adipocytes were analyzed 8 h later by fluorescence microscopy. (G) Representative single-plane image of transfected 3T3-L1 cells is shown. (H) Quantification of the FAM-positive cells relative to the total number of cells. Data is expressed as percentage of cells containing fluorescent si-RNA. Statistical analysis was performed using Mann-Whitney test, ****p < 0.0001. Abbreviations: si-SCR = scrambled siRNA; SEM = standard error of the mean; qRT-PCR = quantitative reverse-transcription polymerase chain reaction; FAM = fluorescein amidite; DAPI = 4′,6-diamidino-2-phenylindole; si-FAM = FAM-labeled si-RNA. Please click here to view a larger version of this figure.
Figure 2: Protein-silencing in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with scrambled si-RNA (si-SCR) or si-RNA against Plin1 (si-PLIN1). (A) Three days after the transfection, mRNA expression of Plin1 was measured by qRT-PCR. The mRNA expression was normalized using 36B4 RNA levels and expressed in arbitrary units, with the si-SCR-treated cells normalized to 1. Results are expressed as the mean + SEM of four independent experiments. Statistical analysis was performed using Student’s t-test, **p < 0.01. (B–C) Three days after the transfection, protein lysates were subjected to western blotting with antibodies directed against PLIN1 and HSP90 (loading control). Representative immunoblots are shown. (C) The amount of PLIN1 was quantified by densitometry scanning analysis and normalized using the amount of HSP90. Data are expressed in arbitrary units, with the si-SCR-treated cells normalized to 1. Results are expressed as the mean + SEM of four independent experiments. Statistical analysis was performed using Student’s t-test, *p < 0.05. (D–F) The 3T3-L1 adipocytes were transfected with si-SCR or si-PLIN1 and plated on coverslips. The expression of PLIN1 was analyzed 96 h later by fluorescence microscopy. (D) Representative single-plane images of 3T3-L1 adipocytes stained with anti-perilipin antibody and an anti-rabbit-Alexa647-conjugated antibody are shown. Scale bars = 10 µm. (E) Three-dimensional (3D) volume-rendering of 3T3-L1 adipocytes segmented in 3D using commercial software. Scale bars = 10 µm. (F) The quantification of PLIN1 signal intensity relative to the total number of cells. Data are expressed in arbitrary units, with the si-SCR-treated cells normalized to 100%. Statistical analysis was performed using Mann-Whitney test, ****p < 0.0001. Abbreviations: si-SCR = scrambled siRNA; si-PLIN1 = siRNA against Plin1; Plin1 = perilipin-1; HSP90 = heat shock protein 90; SEM = standard error of the mean; qRT-PCR = quantitative reverse-transcription polymerase chain reaction; FAM = fluorescein amidite; DAPI = 4′,6-diamidino-2-phenylindole; IB = immunoblotting. Please click here to view a larger version of this figure.
Figure 3: micro-RNA overexpression in 3T3-L1 adipocytes. 3T3-L1 adipocytes were transfected with control micro-RNA mimic (miR-control) or micro-RNA 34a mimic (miR-34a). Three days after the transfection, the cells were harvested for (A) RNA extraction or (B) preparation of protein lysates. (A) The expression of miR-34a was measured by qRT-PCR. The miR expression was normalized using U6 small RNA levels and expressed in arbitrary units, with the miR-control-treated cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed using Student’s t-test, *p < 0.05. (B) Protein lysates were subjected to western blotting with antibodies directed against VAMP2 and TUBULIN (loading control). Representative immunoblots of three independent experiments are shown. (C) The amount of VAMP2 was quantified by densitometry scanning analysis and normalized using the amount of TUBULIN. Data are expressed in arbitrary units, with the miR-control cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed by Student’s t-test, *p < 0.05. Abbreviations: SEM = standard error of the mean; qRT-PCR = quantitative reverse-transcription polymerase chain reaction; VAMP2 = vesicle-associated membrane protein 2; IB = immunoblotting. Please click here to view a larger version of this figure.
Figure 4: Effects of protein or micro-RNA modulation in 3T3-L1 adipocyte function. (A) 3T3-L1 adipocytes were transfected with si-SCR or si-PLIN1. The medium was changed 24 h after the transfection and then collected 48 h later to measure basal lipolysis. Results are expressed as glycerol released in the media (µg/mL), and as the mean + SEM of four independent experiments. Statistical analysis was performed using Student’s t-test, ***p < 0.001. (B) 3T3-L1 adipocytes were transfected with miR-control or miR-34a. The medium was changed 24 h after the transfection, and then, 48 h later, the medium was changed to the depletion medium (DMEM without pyruvate, 25 mM glucose, 1% penicillin and streptomycin, and 0.5% BSA) for 6 h. Then, the cells were treated with 0.5 nM insulin for 5 min. Cells were harvested to prepare protein lysates for western blotting with antibodies directed against phospho-PKB and PKB (loading control). Representative immunoblots of three independent experiments are shown. (C) The amount of phospho-PKB was quantified by densitometry scanning analysis and normalized using the total amount of PKB. Data are expressed in arbitrary units, with the miR-control cells treated with insulin normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed using the two-way ANOVA test, *p < 0.05 compared to miR-control cells treated with insulin. (D) 3T3-L1 adipocytes were transfected with miR-control or miR-34a. The media was changed 24 h after the transfection, and then, 48 h later, the medium was changed to the depletion medium for 6 h. Then, the cells were treated with 0.5 nM insulin for 20 min. Uptake of (2-3H)deoxyglucose was measured over 3 min. Data are expressed in arbitrary units, with the basal glucose uptake in miR-control-treated cells normalized to 1. Results are expressed as the mean + SEM of three independent experiments. Statistical analysis was performed using the two-way ANOVA test, *p < 0.05 compared to miR-control cells treated with insulin. Abbreviations: si-SCR = scrambled siRNA; si-PLIN1 = siRNA against Plin1; SEM = standard error of the mean; PKB = protein kinase B; p-PKB = phosphor-PKB; miR-CTL = miR-control; DMEM = Dulbecco’s modified Eagle’s medium; BSA = bovine serum albumin; ANOVA = analysis of variance; IB = immunoblotting. Please click here to view a larger version of this figure.
| Per well (12-well plate) | Per well (24-well plate) | |
| Oligonucletides | 20 µL | 10 µL |
| (si-RNA, miR…) | ||
| Improved Minimal Essential Medium | 20 µL | 10 µL |
| Transfection reagent | 5.6 µL | 2.8 µL |
| Improved Minimal Essential Medium | 154.4 µL | 77.2 µL |
| Total volume of transfection mix | 200 µL | 100 µL |
Table 1: Transfection reagents required for 12-well and 24-well plate formats.
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Discussion
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 utility of this technique for gain-of-function studies. Although murine cell lines, including the 3T3-L1 cell line, have been typically used to study adipocyte function in vitro, micro-RNA expression patterns and activities in murine tissue are often different from those observed in humans; this is also the case with primary cells and cell lines. Moreover, the medium used for the differentiation of 3T3-L1 fibroblast into adipocytes requires an unphysiological hormone cocktail (insulin, dexamethasone, IBMX, and rosiglitazone), and the differentiated cells are morphologically distinct from in vivo mature adipocytes: they present multilocular lipid droplets instead of a unilocular lipid droplet. This could explain some differences in gene expression and cellular responses between in vitro and in vivo studies.
One benefit of using this reverse-transfection protocol compared to electroporation is that this method is cheaper. Indeed, the reagents are less expensive, and the high efficiency of the reverse transfection decreases the quantities of oligonucleotides needed, and there is no need of expensive equipment such as an electroporator. Moreover, using a lower concentration of siRNA is beneficial as it avoids off-target effects. Furthermore, this reverse-transfection is an easy, fast, gentle, and straightforward method of transfection that requires fewer cells and will ensure excellent cell viability and more robust data compared to electroporation. Although the procedure detailed above has been optimized for the differentiation and reverse-transfection of mouse 3T3-L1 cells, human preadipocytes can also be differentiated into adipocytes5 and easily subjected to reverse-transfection using this protocol. This report shows that adipocytes remain viable, healthy, and responsive to insulin after the reverse-transfection using a new generation of a non-liposomal cationic amphiphile transfection reagent. The reverse-transfection could also be performed using other popular transfection reagents. However, the amounts of oligonucleotides and transfection reagent would need to be optimized to ensure good modulation of expression and no adverse effects on cell viability.
This protocol facilitates the study of the role of proteins and micro-RNAs in adipocyte function, but it could also be used to modulate other non-coding RNAs such as lnc-RNAs, Y-RNAs, or eRNAs. There are critical steps in the protocol that can impact the efficiency of the procedure. The differentiation of 3T3-L1 fibroblasts into adipocytes should be carefully monitored. It is important to reach a high level of differentiation to avoid the proliferation of remaining fibroblasts after the transfection, which would bias the results of the experiment. Another important point is to perform the transfection on newly differentiated mature adipocytes; the best timing is 7-8 days after the beginning of the differentiation protocol, which corresponds to 3-4 days after removing the differentiation cocktail. This will ensure robust transfection efficacy and favor good reattachment of the adipocytes to the plate. Finally, the treatment of adipocytes with trypsin should be monitored carefully to ensure detachment of the adipocytes without damaging the cells.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
This work was supported by INSERM, the Université Côte d'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 Société Francophone du Diabète (SFD), the Association Française d'Etude et de Recherche sur l'Obésité (AFERO), the Institut Thématique Multi-Organismes Technologies pour la Santé (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 Médicale (Equipe FRM, DEQ20180839587). We also thank the Imaging Core Facility of C3M funded by the Conseil Départemental des Alpes-Maritimes and the Région PACA, which is also supported by the GIS IBiSA Microscopy and Imaging Platform Côte d'Azur (MICA).
Materials
| Name | Company | Catalog Number | Comments |
| 12 well Tissue Culture Plate | Dutscher | 353043 | |
| 2.5% Trypsin (10x) | Gibco | 15090-046 | diluted to 5x with D-PBS |
| 2-Propanol | Sigma | I9516 | |
| 3-Isobutyl-1-methylxanthine | Sigma-Aldrich | D5879 | |
| Accell Non-targeting Pool | Horizon Discovery | D-001910-10-05 | |
| Bovine Serum Albumin (BSA) | Sigma | A7030 | |
| Collagen type I from calf skin | Sigma-Aldrich | C8919 | |
| Dexamethasone | Sigma-Aldrich | D1756 | |
| D-PBS | Gibco | 14190144 | |
| Dulbecco's Modified Eagles's Medium (DMEM) | Gibco | 41965062 | 4.5 g/L D-Glucose; L-Glutamine; no Pyruvate |
| Ethanol | Sigma | 51976 | |
| FAM-labeled Negative Control si-RNA | Invitrogen | AM4620 | |
| Fetal Bovine Serum | Gibco | 10270-106 | |
| Free Glycerol Reagent | Sigma-Aldrich | F6428 | |
| Glycerol Standard Solution | Sigma-Aldrich | G7793 | |
| HSP90 antibody | Santa Cruz | sc-131119 | Dilution : 0.5 µg/mL |
| Improved Minimal Essential Medium (Opti-MEM) | Gibco | 31985-047 | |
| Insulin, Human Recombinant | Gibco | 12585-014 | |
| miRIDIAN micro-RNA mimics | Horizon Discovery | ||
| miRNeasy Mini Kit | Qiagen | 217004 | |
| miScript II RT Kit | Qiagen | 218161 | |
| miScript Primer Assays Hs_RNU6-2_11 | Qiagen | MS00033740 | |
| miScript Primer Assays Mm_miR-34a_1 | Qiagen | MS00001428 | |
| miScript SYBR Green PCR Kit | Qiagen | 219073 | |
| Newborn Calf Serum | Gibco | 16010-159 | |
| Oil Red O | Sigma | O0625 | |
| ON-TARGETplus Mouse Plin1 si-RNA SMARTpool | Horizon Discovery | L-056623-01-0005 | |
| Penicillin and Streptomycin | Gibco | 15140-122 | |
| Perilipin-1 antibody | Cell Signaling | 3470 | Dilution : 1/1000 |
| Petri dish 100 mm x 20 mm | Dutscher | 353003 | |
| PKB antibody | Cell Signaling | 9272 | Dilution : 1/1000 |
| PKB Phospho Thr308 antibody | Cell Signaling | 9275 | Dilution : 1/1000 |
| Rosiglitazone | Sigma-Aldrich | R2408 | |
| Transfection reagent (INTERFERin) | Polyplus | 409-10 | |
| α-tubulin antibody | Sigma aldrich | T6199 | Dilution : 0.5 µg/mL |
| Vamp2 antibody | R&D Systems | MAB5136 | Dilution : 0.1 µg/mL |
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