Download Supporting information for Dynamic subcellular localization of

Supporting information for
Dynamic subcellular localization of aquaporin-7 in white adipocytes
Takayuki Miyauchi†, Hiroyuki Yamamoto, Yoichiro Abe, Go J Yoshida,
Aleksandra Rojek, Eisei Sohara, Shinichi Uchida, Søren Nielsen, and
Masato Yasui†
†Authors
for correspondence (e-mail: [email protected]; [email protected])
This file includes:
S1. Materials and Methods
S2. Associated references
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S1. SI Materials and Methods
S1.1. Preparation of mouse white adipose tissue
All procedures relating to the care and treatment of animals were approved
by the animal resource committee of Keio University (No. 12117). The
generation of AQP7-null mice has been described previously [18]. AQP7 +/+
C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). Mice
were anesthetised with isoflurane and euthanised by cervical dislocation.
The white adipose tissues isolated from inguinal tissues of wild type and
AQP7 null-mice at various ages (1 day through 10 weeks) were cut into small
pieces (less than 2 mm thick) and utilized for immunofluorescent imaging.
To compare the localization of AQP7 between lipogenic and lipolytic
conditions, the pieces of tissue were isolated from 3-day-old mice, which still
possessed some multilocular LDs (although larger unilocular LDs were also
beginning to form) and could be more easily isolated than the tissues of
1-day-old mice due to
their larger size. The small pieces of tissue were
exposed to HEPES-buffered DMEM-F12 (11039; Life technologies, Carlsbad,
CA) medium containing 1 μM insulin supplemented with 10% calf serum or
10 μM norepinephrine and incubated for 3 hours in a culture shaker (100
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rpm, 37°C; GBR-200; Taitec, Koshigaya, Japan) for aeration. LDs, nuclei,
and plasma membranes were labeled by a 15-minute incubation with
BODIPY 493/503 (D3922; Life technologies), Hoechst 33342 (H1399; Life
technologies), and a 5-minute incubation with Alexa 488-cholera toxin B
(CT-b)(C-34775; Life technologies), respectively. Prior to fixation, cell
viability was monitored by ensuring that calcein-AM (Calcein_Red-Orange,
C34851; Life technologies) incorporation was greater than 95%.
S1.2. Cell culture, differentiation, and transfection
3T3-L1 preadipocytes (JCRB9014; Health Science Research Resources Bank
of the Japan Health Sciences Foundation, Osaka, Japan) were grown in
Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf
serum, 100 µg/ml streptomycin, and 100 units/ml penicillin at 37°C in a 5%
CO2 incubator, and differentiated into adipocytes as described by Jain et al
[19]. Briefly, 3T3-L1 preadipocytes were incubated with induction medium
containing 10% FBS/DMEM with 0.5 mM isobutylmethylxanthine, 1 µM
dexamethasone, and 10 µg/ml insulin (Sigma, St. Louis, MO). Two days after
induction, the medium was replaced with 10% FBS/DMEM supplemented
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with 1 µg/ml insulin and 10% FBS/DMEM after another 2 days. 3T3-L1
adipocytes were transfected with expression plasmids by electroporation
(Gene Pulsar; BioRad, Hercules, CA) on the 8th day after the induction of
differentiation, incubated for 2 days, and then analyzed by microscopic
imaging.
S1.3. Immunohistochemistry and immunocytochemistry
The small pieces of adipose tissue and 3T3-L1 adipocytes were fixed using
10% trichloroacetic acid (TCA) as described by Hayashi et al [20]. Briefly, a
10% TCA solution was mixed from a stock TCA solution (208-08081; Wako,
Osaka, Japan) just before use. The samples were immersed in ice-cold 10%
TCA for 30 minutes (tissue) or 15 minutes (3T3-L1 adipocytes). The samples
were then permeabilized using 0.2% Triton X-100/G-PBS for 30 minutes at
room temperature. The samples were washed and soaked in blocking
solution (G-PBS containing 10% calf serum and 1% bovine serum albumin),
incubated with primary antibodies for 1 hour, and then labeled with the
appropriate fluorescent secondary antibodies. The characterization of the
affinity-purified anti-mouse AQP7 antibody (1246) has been described
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previously [21]. Alexa Fluor 488-, 555- or 647-conjugated goat anti-rabbit
secondary antibodies were purchased from Molecular Probes (Life
technologies). The samples were washed with G-PBS at least 3 times and
then observed using confocal microscopy. For microscopic imaging, the small
pieces of adipose tissue were transferred to glass-bottom dishes (3910 and
3911; Iwaki, Asahi techno glass, Japan) and immobilised using a glass
coverslip.
S1.4. Imaging
For immunohistochemical and live cell fluorescence imaging, a FV1000
confocal microscope (Olympus, Tokyo, Japan) equipped with an inverted
microscope (IX-81) and a 60×/1.20 NA water-immersion objective lens
(UPlanSApo 60XW) were used. Imaging using coherent anti-Stokes Raman
scattering (CARS) microscopy was performed as described previously [22].
For the observation of LDs, CARS microscopy was used with modification for
the lipid CH2 symmetric stretch vibration (Raman shift: 2,845 cm-1, pump:
816.7 nm, Stokes: 1064 nm). Briefly, the CARS microscope was set up based
on the FV1000 microscope equipped with the 60×/1.20 NA water-immersion
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objective. Forward-CARS signals were corrected via a custom-built forward
detector via a condensing lens (IX2-LWUCD, NA0.55; Olympus). For live
imaging of lipolysis, the cells were maintained in HEPES-buffered
DMEM-F12 (2% FBS) at 37°C using an imaging chamber (Tokai hit,
Fujinomiya, Japan) for the duration of the time-lapse capture. 3T3-L1
adipocytes cultured in glass-bottom dishes were stimulated by
bath-application of 5 µM forskolin or 10 µM norepinephrine. Image
calculations were performed on background-subtracted channel images. The
images were analysed using FluoView software (ver. 2.01; Olympus), and
Metamorph software (ver. 7.7; Molecular devices, Downingtown, PA).
S1.5. Plasmid construction
Mouse AQP7 (NM_007473) was inserted into pEGFP-N1 (Clontech),
pVenus-N1 [38], and pmCherry-N1 [39] at the EcoRI and SmaI restriction
sites. Mouse CGI-58 (NM_026179) was inserted into pECFP-C1 using XhoI
and BamHI, as described by Yamaguchi et al. [28], generating an N-terminal
fluorescent protein (FP)–CGI-58 fusion. Mouse FSP27 (NM_178373) was
inserted into pEGFP-N1 using SalI and BamHI, as described by Keller et
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al.[40], generating a FSP27–C-terminal FP- fusion. Mouse perilipin 1
(NM_175640) was inserted into pECFP-N1 and pmCherry-N1 using the
NheI and AgeI sites, as described by Moore et al.[41], generating a perilipin
1–C-terminal FP fusion. Mouse HSL cDNA was purchased from Life
Technologies (Full-Length Mammalian Gene Collection; clone ID 4188567).
Mouse ATGL cDNA was also purchased from Life Technologies (clone ID
30024535). The integrities of all plasmids were verified by DNA sequencing.
S1.6. Oligonucleotide primers
The cDNAs of AQP7, perilipin 1, FSP27, and CGI-58 were PCR-amplified
from cDNA of differentiated 3T3-L1 adipocytes using following primer sets:
AQP7, 5’-GAATTCACCATGGCCCCCAGGTCCGTGCTG-3’ /
5’-ACCTAGGTAGGCGCCCAGAAG-3’ and
5’-CTTCCTGGATGAGGCATTCGTG-3’ / 5’GATATCTGAAGTGCTCTAGAGGCACAGAG-3’; CGI-58,
5’-ACTCGAGCTAAAGCGATGGCGGCGGAGGAG-3’ /
5’-GGATCCTCAGTCTACTGTGTGGCAGATCTC-3’; FSP27, 5’AGTCGACACCATGGACTACGCCATGAAGTC-3’ / 5’7
GGATCCCGTTGCAGCATCTTCAGACAGGC-3’; perilipin 1,
5’-ACGCTAGCGACCATGTCAATGAACAAGGG-3’ / 5’CCCCGGACCCTTTGCGCTCCGCCTCTGCTG-3’ and
5’-AACGTGGTAGACACTGTGGTACACTATGTG-3’ / 5’ACCGGTGGGCTCTTCTTGCGCAGCTGGCTG-3’. RT-PCR for the
quantification of mRNA expressed in 3T3-L1 cells was performed using
following primer sets: aqp7, 5’-CTGCTGCTTCAGGTCCA-3’ /
5’-GAATGCCTCATCCAGGA-3’; -actin, GAGCACAGCTTCTTTGCA-3’ /
5’-TCACAATGCCTGTGGTA-3’.
S1.7. Data analysis
Statistical analysis was performed using KaleidaGraph (ver. 4.1; Synergy
software), and Excel (Microsoft) with the Statcel2 add-in (OMS publishing,
Tokorozawa, Japan). Normally distributed variables were compared using
Student’s t-test, unequally distributed variances using Welch's t-test, and
nonnormally distributed variables using the Mann-Whitney U-test.
Statistical significance was defined as p < 0.05. The results are
representative of more than three separate experiments.
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S2. Associated reference
38.
Nagai T, et al. (2002) A variant of yellow fluorescent protein with fast
and efficient maturation for cell-biological applications. Nat. Biotechnol.
20:87-90.
39.
Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ (2006)
Novel chromophores and buried charges control color in mFruits.
Biochemistry (Mosc). 45:9639-9647.
40.
Keller P, et al. (2008) Fat-specific protein 27 regulates storage of
triacylglycerol. J. Biol. Chem. 283:14355-14365.
41.
Moore HP, Silver RB, Mottillo EP, Bernlohr DA, Granneman JG
(2005) Perilipin targets a novel pool of lipid droplets for lipolytic attack by
hormone-sensitive lipase. J. Biol. Chem. 280:43109-43120.
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