High efficiency lipid-based siRNA transfection of adipocytes in suspension.

Abstract

Fully differentiated adipocytes are considered to be refractory to introduction of siRNA via lipid-based transfection. However, large scale siRNA-based loss-of-function screening of adipocytes using either electroporation or virally-mediated transfection approaches can be prohibitively complex and expensive.
We present a method for introducing small interfering RNA (siRNA) into differentiated 3T3-L1 adipocytes and primary human adipocytes using an approach based on forming the siRNA/cell complex with the adipocytes in suspension rather than as an adherent monolayer, a variation of "reverse transfection".
Transfection of adipocytes with siRNA by this method is economical, highly efficient, has a simple workflow, and allows standardization of the ratio of siRNA/cell number, making this approach well-suited for high-throughput screening of fully differentiated adipocytes.

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High Efficiency Lipid-Based siRNA Transfection of
Adipocytes in Suspension
Gail Kilroy1, David H. Burk2, Z. Elizabeth Floyd1*
1Ubiquitin Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana, United States of America, 2Cell Biology and Bioimaging Core,
Pennington Biomedical Research Center, Baton Rouge, Louisiana, United States of America
Abstract
Background: Fully differentiated adipocytes are considered to be refractory to introduction of siRNA via lipid-based
transfection. However, large scale siRNA-based loss-of-function screening of adipocytes using either electroporation or
virally-mediated transfection approaches can be prohibitively complex and expensive.
Methodology/Principal Findings: We present a method for introducing small interfering RNA (siRNA) into differentiated
3T3-L1 adipocytes and primary human adipocytes using an approach based on forming the siRNA/cell complex with the
adipocytes in suspension rather than as an adherent monolayer, a variation of ‘‘reverse transfection’’.
Conclusions/Significance: Transfection of adipocytes with siRNA by this method is economical, highly efficient, has a simple
workflow, and allows standardization of the ratio of siRNA/cell number, making this approach well-suited for high-
throughput screening of fully differentiated adipocytes.
Citation: Kilroy G, Burk DH, Floyd ZE (2009) High Efficiency Lipid-Based siRNA Transfection of Adipocytes in Suspension. PLoS ONE 4(9): e6940. doi:10.1371/
journal.pone.0006940
Editor: Aimin Xu, University of Hong Kong, China
Received May 21, 2009; Accepted August 7, 2009; Published September 11, 2009
Copyright: ? 2009 Kilroy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by COBRE NIH P20 RR02195 (ZEF) from the National Institutes of Health. The Cell Biology and Bioimaging(DHB) and Genomics
Cores are supported in part by COBRE (NIH P20-RR021945) and CNRU (NIH 1P30-DK072476) center grants from the National Institutes of Health. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Elizabeth.Floyd@pbrc.edu
Introduction
Murine 3T3-L1 adipocytes are a well-characterized cell culture
model that is widely used to study the role of adipocyte biology in
obesity and type 2 diabetes. These properties make 3T3-L1
adipocytes an attractive model for carrying out loss-of-function
assays using siRNA technology. However, fully differentiated 3T3-
L1 adipocytes are among the most difficult cell types to transfect
efficiently with siRNA using standard lipid-based techniques.
Typically, siRNA is introduced into 3T3-L1 adipocytes using
either electroporation or virally-mediated approaches [1,2,3,4].
Both of these approaches have limitations in systematic siRNA-
mediated screening experiments, including the potential cell
damage and equipment and reagent costs associated with
electroporation in a high-throughput format or the complexity
and safety issues associated with virally-mediated transfection.
Alternatives include peptide-based transfection reagents that are
highly efficient, but require sonication of the peptide prior to
transfection and have not been demonstrated in fully differentiated
adipocytes [5]. ‘‘Reverse transfection’’, also known as solid phase
optimized transfection RNAi (SPOT-RNAi) [6], is an alternative
that uses glass plates or cell culture plates preloaded with siRNA
and to which the cells of interest are then added.
With improved transfection efficiency, lipid-based siRNA
transfection using a version of ‘‘reverse transfection’’ in which
the siRNA and cells are mixed in suspension would offer the
simplest and least expensive approach to systematic screening
using siRNA in adipocytes. The adipocytes would then be allowed
to reattach to an adherent plate surface while in the presence of
the siRNA complex. This approach has been reported in the
human melanoma cell line LOX, another cell line that is
considered difficult to transfect using lipid-based reagents [7].
Herein, we present a method for lipid-mediated siRNA
transfection of fully differentiated 3T3-L1 adipocytes and primary
human adipocytes that is based on incubating the siRNA/lipid
complex with the detached adipocytes in suspension. This results
in highly efficient siRNA transfection and is simple, cost-effective,
and nontoxic, making this approach well suited to systematic high
throughput siRNA screening of fully differentiated adipocytes.
Materials and Methods
Materials
Dulbecco’s Modified Eagle’s Media (DMEM) was purchased
from MediaTech. Bovine and fetal bovine (FBS) serums were
obtained from Hyclone. Insulin, IBMX, dexamethasone , DAPI,
and collagen (#C-7116) were purchased from Sigma-Aldrich.
OptiMEM, Calcein-AM, and propidium iodide were from
Invitrogen. The mouse monoclonal PPARc (sc-7273), goat
polyclonal lamin A/C (sc-6215), and mouse monoclonal b-actin
(sc-47778) antibodies were purchased from Santa Cruz Biotech-
nology. The rabbit polyclonal E6-AP antibody (A300–352A) was
from Bethyl Laboratories. Horseradish peroxidase conjugated
secondary antibodies were obtained from Jackson Immunore-
search Laboratories. DharmaFECT transfection reagents, siGLO
RISC-free labeled with DY-547 (Rhodamine filter) siRNA,
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siGenome non-targeting siRNA Pool #2, lamin A/C siRNA,
TBL-1 and TBLR-1 siRNA were purchased from Thermo Fisher
Scientific-Dharmacon. E6-AP and PPARc siRNAs were pur-
chased from Santa Cruz Biotechnology. Polyfect was purchased
from Qiagen and Xfect was purchased from Clontech. All
TaqMan primer/probes pairs were obtained from Applied
Biosystems. Differentiated human primary adipocytes (lot #
L041806) were purchased from Zen-Bio, Inc.
Cell Culture
Murine 3T3-L1 preadipocytes were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) high glucose containing 10%
calf serum and antibiotics (100 units/ml penicillin G and 100 mg/
mlstreptomycin).To obtainfullydifferentiatedadipocytes,the3T3-
L1 preadipocytes were plated and grown on 10 cm plates to 2 days
post confluence and induced to differentiate using a standard
induction cocktail of 3-isobutyl-1-methylxanthine, dexamethasone,
and insulin (MDI) as previously described [8]. After 48 hours this
mediumwasreplaced withDMEMhighglucose supplemented with
10% fetal bovine serum (FBS) and the cells were maintained in this
medium. The 3T3-L1 adipocytes were used for transfection at day
4-6 post-induction when lipid droplets were readily apparent.
The differentiated adipocytes were rinsed in phosphate-buffered
saline (PBS, 5 ml) prewarmed to 37uC and detached from the
plate via trypsin treatment (1 ml of 0.25% trypsin/10 cm plate) at
37uC. The adipocytes were in contact with the trypsin only until
the cells began to detach, approximately 2–5 minutes. The
detached adipocytes were gently resuspended in DMEM, high
glucose with 10% FBS and antibiotics (5 ml) and collected by
centrifugation at 5146g (1000 rpm, Beckman Coulter GH 3.8
rotor) for five minutes at room temperature. The pelleted
adipocytes were gently resuspended in DMEM, high glucose with
10% FBS and antibiotics (5 ml) and an aliquot was counted using
a hemocytometer after the addition of trypan blue. A typical yield
was 1.021.26107cells/plate.
Optimization of siRNA Transfection of 3T3-L1 Adipocytes
To determine optimal conditions for lipid-based siRNA
transfection of the adipocytes, we established a grid in the 48
well format to test four variables concurrently: cell density,
transfection reagent, siRNA concentration, and transfection
reagent volume. Each grid allows optimization using two cell
densities, two concentrations of siRNA, and four different
transfection reagents at three volumes each. The plates were
collagen-coated throughout these experiments. The adipocytes
were replated at 5.46104cells/cm2or 1.166105cells/cm2in the
48 well format at 1 cm2/well. We initially used several lipid-based
transfection reagents including DharmaFECT 4, DharmaFECT
Duo, Polyfect, and Xfect. Each transfection reagent was tested at
0.7 ml/cm2, 1.4 ml/cm2, or 3.7 ml/cm2. At 3.7 ml/cm2of the
transfection reagents, the cells detached from the plate within
24 hours. Therefore subsequent experiments were carried out with
DharmaFECT D4, DharmaFECT Duo, Polyfect, or Xfect at
0.7 ml/cm2or 1.4 ml/cm2with optimal results obtained using
DharmaFECT Duo at 1.4 ml/cm2. During optimization, fluores-
cently labeled siRNA that does not induce the RNA silencing
complex (RISC-free) was used at 25 nM or 100 nM for the siRNA
complex. The siRNA complex was formed in the 48 well plate by
adding equal volumes of the stock concentration of siRNA and
OptiMEM and incubating at room temperature in a laminar flow
hood. At the end of 5 minutes, the transfection reagent was added
along with additional OptiMEM to yield a final total volume of
40 ml/well and incubated at room temperature. At the end of 20
minutes, the resuspended adipocytes at the desired concentration
in a total volume of 200 ml were added and the siRNA complex:
adipocytes mixture was plated and incubated at 37uC, 5% CO2
for 24 hours before the media was exchanged for DMEM, high
glucose with 10% FBS and antibiotics.
Detection of Fluorescent Labeled siRNA and DAPI
Stained Nuclei
At 24 and 48 hours post-transfection, transfection efficiency was
assayed via fluorescent detection of the labeled siRNA using a
Nikon Eclipse TS100 inverted microscope equipped with
rhodamine and DAPI filters and Metamorph version 6.1 software.
DAPI staining was used to determine the cellular location of the
transfected siRNA. Adipocytes transfected with fluorescent labeled
[DY-547 (Rhodamine filter)] RISC-free siRNA were incubated
with DAPI (0.14 mM) in media for 2.5 hours at 37uC, 5% CO2
without fixation of the cells. Thereafter, the adipocytes were rinsed
twice with media and the DAPI signal was visualized. Merged
images were generated using ImageJ software.
Classical Transfection of 3T3-L1 adipocytes with siRNA
3T3-L1 preadipocytes were plated at 1.1656105cells/cm2in a
48 well plate format and induced to differentiate 24 hours later
using the standard induction cocktail as described under Cell
Culture. The adipocytes were transfected with 25 nM or 100 nM
siRNA and 1.4 ml/cm2DharmaFECT Duo for each well at days 4
or 5 post-induction. Transfection efficiency was assessed 48 hours
post-transfection by detection of fluorescent labeled RISC-free
siRNA and western blot analysis of siRNA targeted proteins.
Transfection of Human Primary Adipocytes with siRNA in
Suspension
Primary human subcutaneous adipocytes were obtained at day 7
post induction and were maintained in DMEM/F-12 with 3% FBS,
33 mM biotin, 17 mM pantothenate, 1 mM bovine insulin, 1 mM
dexamethasone, and 100 U penicillin/100 mg streptomycin/
0.25 mg Fungizone. At day 9 post-induction, the adipocytes were
transfected in suspension as described for the 3T3-L1 adipocytes
using 1.1656105cells/25 nM siRNA or 1.1656105cells/100 nM
siRNA and plated on collagen-coated 48 well plates at
4.16104cells/cm2. Transfection efficiency was assessed 48 hours
post-transfection by detection of fluorescent labeled RISC-free
siRNA and western blot analysis of siRNA targeted proteins.
Cell Viability Assay
Cell viability was determined using calcein-acetoxymethyl
(calcein-AM) to stain viable cells and DNA intercalation of
propidium iodide (PI) to stain dead cells. The 3T3-L1 adipocytes
at 5.46104or 1.166105cells/cm2were transfected with non-
targeting siRNA (25 nM and 100 nM of siGenome non-targeting
siRNA, pool #2, which includes luciferase siRNA) and incubated
for 48 hours with a single media change at 24 hours after
transfection. The cells were then rinsed twice with PBS and
incubated with calcein-AM (10 mM) and PI (10 mM) in PBS at
37uC for 15 minutes. The fluorescent signals were detected and
quantitated using a Flexstation 2 fluorometer (Molecular Devices)
and Softmax Pro 4.8 software at an excitation of 490 nm and
emission of 515 nm for detection of fluorescence generated from
the calcein-AM and an excitation of 535 nm and emission of
617 nm for detection of PI bound to DNA.
Real-time RT-PCR
Total RNA was purified from cultured cells using TriReagent
(Molecular Research Center) according to the manufacturer’s
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instructions. When comparing adipocyte marker genes pre and
post-replating (Figure 1B) with expression of the marker genes in
the preadipocytes, total RNA was purified from equivalent cell
numbers harvested prior to replating (‘‘Pre’’) and 48 hours after
replating (‘‘Post’’). RNA (200 ng) was reverse transcribed using
Multiscribe Reverse Transcriptase (Applied Biosystems) with
random primers at 37uC for 2 hour. Real-time PCR was
performed with TaqMan chemistry (Applied Biosystems) using
the 7900 Real-Time PCR system (Applied Biosystems) and
universal cycling conditions (50uC for 2 minutes; 95uC for 10
minutes; 40 cycles of 95uC for 15 seconds and 60uC for 1 minute;
followed by 95uC for 15 seconds, 60uC for 15 seconds and 95uC
for 15 seconds). All results were normalized to a Cyclophilin B
expression control.
Oil Red O staining
Oil Red O staining was performed as described by Green and
Kehinde [9].
Preparation of Whole Cell Extracts
Cell monolayers were rinsed with phosphate-buffered saline
(PBS) and harvested in a denaturing buffer containing 50 mM
Tris-Cl, pH 7.4 with 150 mM NaCl, 1 mM EDTA, 1% Igepal,
0.5% Na-deoxycholate, 0.1% SDS, protease inhibitors (1 mM
PMSF, 1 mM pepstatin, 50 trypsin inhibitory milliunits of
aprotinin, 10 mM leupeptin). Protein concentrations of whole cell
extracts were determined using a BCA assay (Thermo Fisher
Scientific, Rockford, IL) according to the manufacturer’s instruc-
tions.
Figure 1. Adipocytes continue to differentiate after harvesting and replating post-induction. The 3T3-L1 preadipocytes were induced to
undergo adipogenesis and harvested at day 3–4 post induction when lipid droplets were clearly visible. (A) Oil Red O staining of neutral lipids forty-
eight hours after harvesting and replating. (B) The gene expression of adipocyte marker genes aP2, LPL, PPARc, and adiponectin was measured via
real-time PCR upon harvesting (Pre) and forty eight hours after (Post) the adipocytes were replated at 5.46104cells/cm2(low) or 1.166105cells/cm2
(high) and compared to the expression of each gene in preadipocytes prior to induction (preAd). Expression of each gene was assayed in triplicate,
normalized to cyclophilin B gene expression, and reported as the mean and standard deviation. The results are representative of experiments carried
out twice independently.
doi:10.1371/journal.pone.0006940.g001
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Gel Electrophoresis and Immunoblotting
Proteins were separated in polyacrylamide (National Diagnos-
tics) gels containing sodium dodecyl sulfate (SDS) according to
Laemmli [10] and transferred to nitrocellulose (BioRad) in 25 mM
Tris, 192 mM glycine, and 20% methanol. Following transfer, the
membrane was blocked in 4% milk for 1 hour at room
temperature. The membranes were incubated with mouse
monoclonal anti-PPARc (1:125 dilution), rabbit polyclonal anti-
lamin A/C (1:200 dilution), or rabbit polyclonal anti-E6-AP
(1:2000 dilution) as indicated for 1–2 hours at room temperature.
Following extensive washes, the results were visualized with
horseradish peroxidase (HRP)-conjugated secondary antibodies
and enhanced chemiluminescence (Pierce).
Results and Discussion
We first confirmed that the fully differentiated adipocytes could
be replated after trypsinization in a 48 well format and continue to
express adipocyte markers. As shown in Figure 1A, when
harvested and replated on day 4 post-induction, the adipocytes
continue to accumulate neutral lipids when assayed using Oil Red
O staining (Figure 1A) 48 hours after replating on the collagen-
coated plates. When replated at 5.86104cells/cm2(‘‘low’’ cell
density, Figure 1B) or 1.166105cells/cm2(‘‘High’’ cell density,
Figure 1B), there is increased gene expression of lipoprotein
lipase (LPL), fatty acid binding protein (aP2), peroxisome
proliferator activated receptor gamma (PPARc), and adiponectin
(Figure 1B) when compared to the levels of each gene present in
the preadipocytes, indicating the adipocytes continue to undergo
differentiation after harvesting and re-adherence to the collagen-
coated plates. LPL and adiponectin were significantly increased at
a cell density of 1.166105cells/cm2(‘‘High’’) compared to a cell
density of 5.86104cells/cm2(‘‘low’’).
We then used a 48 well grid (Figure 2A) to test a combination
of factors, including cell density, transfection reagent, transfection
reagent volume, siRNA concentration, and incubation time, to
determine if the adipocytes could be efficiently transfected in
suspension using lipid-based reagents. We initially assessed the
fluorescence signal obtained with each transfection reagent
(Figure 2B). Maximal fluorescent signal/cell number plated was
obtained with three transfection reagents (D4, Duo, Xfect) at
either 25 nM siRNA (D4 or Xfect) or 100 nM siRNA (Duo). The
optimal combination of transfection efficiency and cell viability in
thefully differentiatedadipocytes
1.166105cells/cm2, 100 nM siRNA, and 1.4 ml/cm2Dharma-
FECT Duo. As shown in Figure 3, the fluorescent-tagged siRNA is
localized in the adipocytes and appears to be excluded from the
lipid droplets. Co-staining with DAPI indicates that the siRNA is
localized to the cytoplasm (Figure 3C).
As an example of our approach, to test the efficiency of
transfection using 1.166105cells/cm2, 1.4 ml of DharmaFECT
Duo/cm2, and 100 nM siRNA, for each well of the 48 well plate,
the siRNA complex was formed by combining 10 ml of OptiMEM
and 10 ml of 2 mM siRNA (in H2O) at room temperature. After 5
minutes, 18.6 ml OptiMEM was added followed by 1.4 ml
DharmaFECT Duo and incubated at room temperature. At the
end of 20 minutes, 200 ml of adipocytes resuspended in media at
5.86105cells/ml was added to obtain 1.166105cells/cm2(for
1 cm2well).
When cell viability under the optimized conditions was assayed
by the production of calcein from calcein-AM (live cells) or PI
binding to DNA (dead cells), the overwhelming majority of
adipocytes remain viable compared to the dead cells as assayed
using fluorescence detection of calcein and DNA bound
propidium iodide (Figure 4). This indicates lipid-based siRNA
transfection of the adipocytes while in suspension does not
adversely affect the viability of fully differentiated adipocytes.
The effectiveness of RNAi experiments is determined by the
efficiency of siRNA transfection and the ability of the siRNA
sequence to silence a specific target mRNA. To test the efficiency
of gene knockdown, we assayed the siRNA-mediated decrease in
expression of the peroxisome proliferator activated receptor
gamma (PPARc), a nuclear receptor that is required for the
formation and maintenance of adipocytes [11]. Along with PPARc
was obtainedusing
Figure 2. Optimization of siRNA transfection of adipocytes in
suspension. (A) Grid layout for testing transfection variables based on
a 48 well plate format. This grid accommodates testing two
concentrations of siRNA (1,2) when transfecting cells at two densities/
cm2(3,4) with four transfection reagents (A,B,C,D) at three concentra-
tions each (E, F, G). (B) Maximal fluorescent signal/cell number plated
was obtained with three transfection reagents (D4, Duo, Xfect) at either
25 nM siRNA (D4 or Xfect) or 100 nM siRNA (Duo). The siRNA is siGLO
RISC-free labeled with DY-547 (rhodamine filter). The cells were plated
at 1.166105cells/cm2. The fluorescent signals were detected and
quantitated using a Flexstation 2 fluorometer (Molecular Devices) and
Softmax Pro 4.8 software. The fluorescent signal was assayed in
triplicate, normalized to the number of cells plated/well, and reported
as the mean and standard deviation from experiments carried out twice
independently.
doi:10.1371/journal.pone.0006940.g002
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as our gene of interest, we also transfected the fully differentiated
adipocytes with siRNA directed against lamin A/C as a control
target and the luciferase siRNA (siRNA Pool #2) as a non-
targeting control. In addition, we are interested in the role of the
ubiquitin proteasome system in regulating PPARc transcriptional
activity in adipocytes. Therefore, we also tested the efficiency of
knockdown of three ubiquitin ligases that regulate nuclear
hormone receptor activity. E6-AP regulates nuclear receptor
activity and targets the estrogen receptor alpha to the proteasome
for degradation [12]. The TBL-1 and TBLR-1 genes are ubiquitin
ligases that target the nuclear receptor corepressor NCoR to the
proteasome for degradation [13,14]. As shown in Figure 5, we
Figure 3. Adipocytes in suspension are efficiently transfected with siRNA. Uptake of the fluorescent-labeled siRNA was assayed at 48 hours
post transfection of adipocytes plated at 1.166105cells/cm2. (A) Brightfield and (B) fluorescent (rhodamine filter) image of the transfected cells taken
with a 40X objective. (C) Co-staining with DAPI and the fluorescent-labeled siRNA indicates the siRNA is located in the cytoplasm. The image in (C) is
from a different experiment than the paired images in (A) and (B). This experiment was carried out independently greater than four times.
doi:10.1371/journal.pone.0006940.g003
Figure 4. 3T3-L1 adipocytes maintain viability with lipid-based siRNA transfection. Cell viability was determined using Calcein-AM (green,
viable cells) and propidium iodide (red, non-viable cells) staining in a ‘‘live-dead’’ assay. The assay was carried out at 48 hours post-transfection using
non-targeting siRNA (Dharmacon siRNA pool #2 containing luciferase siRNA). (A) Brightfield images taken with a 20X (upper panel) and 40X objective
(lower panel). (B) Fluorescent images taken with a 20X (upper panel) and 40X objective (lower panel) showing minimal PI staining. The experiment
was carried out twice independently.
doi:10.1371/journal.pone.0006940.g004
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Figure 5. Specific genes are efficiently targeted with lipid-based siRNA transfection of adipocytes. Knockdown of specific genes was
assayed at 48 hours post-transfection using either the RISC-free control siRNA (control), nontargeting siRNA (NT), or siRNA targeted to the indicated
gene (target). (A) Thirty-five mg of protein was loaded in each lane and separated by SDS-PAGE. Knockdown of lamin A/C, PPARc1 and PPARc2, and
E6-AP was assayed via western blot analysis. Equal loading of each lane was determined using b-actin expression. (B) knockdown of TBL-1 and TBLR-1
was assayed via real-time PCR in triplicate and reported as the mean and standard deviation. These experiments were carried out independently
greater than four times.
doi:10.1371/journal.pone.0006940.g005
Figure 6. Transfection of adherent 3T3-L1 adipocytes with siRNA does not decrease expression of targeted proteins. Forty-eight
hours post-transfection, transfection efficiency was assayed as uptake of fluorescent-labeled siRNA and the siRNA-mediated effect on targeted
protein expression. (A) Brightfield and fluorescent (rhodamine filter) images of the transfected cells were taken with a 40X objective. (B) Knockdown
of lamin A/C and PPARc was assayed via western blot analysis post-transfection with either the RISC-free control siRNA (control), nontargeting siRNA
(NT), or siRNA targeted to the indicated gene (target). Thirty-five mg of protein was loaded in each lane and separated by SDS-PAGE. Equal loading of
each lane was determined using b-actin expression. The experiment was carried out twice independently.
doi:10.1371/journal.pone.0006940.g006
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Figure 7. Transfection of human primary adipocytes in suspension with siRNA is associated with decreased expression of targeted
proteins. The human adipocytes were transfected on day 9 post-induction (1.1656105cells/100 nM siRNA ) and plated at 4.16104cells/cm2. Forty-
eight hours post-transfection, transfection efficiency was assayed as uptake of fluorescent-labeled siRNA and the siRNA-mediated effect on targeted
protein expression. (A) Brightfield and fluorescent (rhodamine filter) images of the transfected cells were taken with a 40X objective. (B) Knockdown
of lamin A/C and PPARc was assayed via western blot analysis post-transfection with either the RISC-free control siRNA (control), nontargeting siRNA
(NT), or siRNA targeted to the indicated gene (target). Thirty-five mg of protein was loaded in each lane and separated by SDS-PAGE. Equal loading of
each lane was determined using b-actin expression. The mean and standard deviation of the ratio lamin A/C and PPARc compared to b-actin was
determined after the expression levels of each protein were quantified using Un-Scan-It software (version 6.1) from samples run in triplicate. The
experiment was carried out twice independently.
doi:10.1371/journal.pone.0006940.g007
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obtain efficient knockdown of lamin A/C, PPARc, and E6-AP
proteins when assayed via western blot analysis (Figure 5A) and
TBL-1 and TBLR-1 when the mRNA levels are assayed via real-
time qRT-PCR (Figure 5B).
To determine if the siRNA transfection conditions established
for adipocytes in suspension would be applicable to adherent
adipocytes, we compared siRNA transfection of the 3T3-L1
adipocytes in suspension with the classical approach of introducing
siRNA into cells that remain attached (Figure 6). While the
fluorescent-tagged RISC-free siRNA is clearly present in the
adipocytes (Figure 6A), there was no decrease in the level of the
targeted proteins, including PPARc as an adipocyte specific target
(Figure 6B). The lack of change in target protein expression is
surprising, given the efficiency of introducing the fluorescent
tagged RISC-free siRNA into the adherent adipocytes. However,
this result suggests that incubation of the siRNA complex with the
adipocytes in suspension or the process of adipocyte readherence
in the presence of a targeting siRNA complex may enhance
induction of the RNAi silencing complex (RISC) response or
delivery of the siRNA to RISC in the adipocytes.
To extend our method to a model of human adipocytes, we
applied the optimized (1.1656105cells/100 nM siRNA) condi-
tions for the 3T3-L1 adipocytes to fully differentiated primary
human adipocytes cultured from subcutaneous adipose tissue
(Figure 7). The fluorescent-tagged siRNA is apparent in the
human adipocytes and is excluded from the lipid droplets
(Figure 7A) as occurs with the 3T3-L1 adipocytes (Figure 3A,B).
Western blot analysis shows that siRNA targeting lamin A/C and
PPARc reduces the expression of both proteins (Figure 7B). While
further optimization is expected to improve the efficiency of gene
knockdown, PPARc protein expression is reduced to approxi-
mately 40% of the control levels and lamin A/C protein
expression is reduced to approximately 20% of the control levels
using the transfection conditions established for the 3T3-L1
adipocytes.
Our results show that lipid-mediated siRNA transfection of fully
differentiated adipocytes occurs in suspension with high efficiency
as determined by localization of the fluorescent-tagged siRNA in
the adipocyte cytoplasm and the decrease in the expression level of
five independent and specific targets, including the adipocyte-
specific PPARc and a small set of ubiquitin ligases. We conclude
that lipid-based siRNA transfection of 3T3-L1 adipocytes and
primary human adipocytes in suspension yields gene knockdown
results that are valid as a model for loss-of-function studies in fully
differentiated adipocytes. While optimization is required for
siRNA-based transfection of any cell type, transfection of
adipocytes with siRNA by this method is economical, highly
efficient, has a simple workflow, and allows standardization of the
ratio of siRNA/cell number, making this approach well-suited for
high-throughput screening of fully differentiated adipocytes. In
these experiments, we used transfection reagents from a limited
number of suppliers, but anticipate that other lipid-based
transfection reagents will also provide efficient siRNA transfection
of the adipocytes in suspension.
Author Contributions
Conceived and designed the experiments: GK ZEF. Performed the
experiments: GK DHB ZEF. Analyzed the data: GK DHB ZEF.
Contributed reagents/materials/analysis tools: DHB ZEF. Wrote the
paper: ZEF.
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Adipocyte siRNA Transfection
PLoS ONE | www.plosone.org8 September 2009 | Volume 4 | Issue 9 | e6940