Supplementary Methods
Animals and Physiological Studies
Animals were maintained on a normal light/dark cycle and handled in accordance
with Stanford University Animal Care and Use Guidelines. Genotyping for all mice was
performed by PCR on tail DNA as described1-4. All blood glucose measurements were
performed on blood isolated from the tail vein using an Elite Glucometer (Bayer, Elkhart,
IN). Random-fed blood glucose measurements were collected between 8 and 10 AM.
Glucose tolerance tests were performed after a 14-hour fast. Mice were injected
intraperitonealy (IP) with D-glucose (2 g/kg body weight), and blood glucose levels were
measured at the indicated times. For insulin secretion studies, mice were injected with Dglucose (3 g/kg body weight) or arginine (2 g/kg body weight), and blood was collected
from the tail vein at the indicated times. Insulin levels were determined by ELISA as
described below.
For serum insulin level determination, mice were euthanized and blood was
collected from the abdominal aorta. Serum was isolated by centrifugation, and insulin
levels were determined using an Ultrasensitive Insulin ELISA kit (Alpco, Windham,
NH). To measure pancreatic insulin and glucagon content, the pancreas tail was isolated,
homogenized in acid alcohol, and extracted overnight at 4˚C. The solution was
centrifuged to remove unhomogenized tissue and neutralized.
Insulin content was determined by ELISA as above, and glucagon levels were
measured by RIA (Linco Research Immunoassay, St. Louis, MO). Intraperitoneal insulin
tolerance tests were performed on random-fed animals between 2 and 4 PM. Mice were
injected IP with 0.75 units/kg body weight of human crystalline insulin (Lilly,
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Indianapolis, IN), and blood glucose measurements were taken at the indicated times and
expressed as a percentage of the initial blood glucose concentration. To assess ß-cell
proliferation, mice were injected IP with BrdU (50 mg/kg body weight), and sacrificed 24
hours after labeling. BrdU was visualized by insulin and BrdU co-staining (see below).
For all Tet-On experiments, Dox was added to the drinking water (1 g/L) and changed 3
times per week.
Islet isolation and experimentation
Islets were isolated by intraductal collagenase (Sigma, St. Louis, MO) perfusion
and ficoll gradient (Cellgro, Herndon, VA) separation from acinar and ductal tissue. Islets
were then handpicked using dithizone (Sigma, St. Louis, MO) detection of zinc granules.
After isolation, islets were either placed in Trizol (Invitrogen, Chicago, IL) for RNA
isolation or in culture media for further experiments. After isolation, islets were placed in
RPMI + 10% heat-inactivated FCS (Hyclone, Logan, UT) and cultured at 37˚C and 5%
CO2. For Tet-on experiments, Dox (1 µg/ml) was added to the growth media to induce
NFATc1nuc expression. Islets were harvested 48 hours after Dox addition for RNA or
protein collection.
For protein isolation, islets were lysed in 50 µl of lysis buffer (50 mM Tris-Cl, pH
8.0, 150 mM NaCl, 0.5% NP-40, 0.1% SDS, 0.5% Deoxycholate, 0.02% Sodium Azide)
with a protease inhibitor cocktail (Calbiochem, San Diego, CA) and protein was
quantified by standard techniques. 50 µg of protein was resolved on a 4-15% SDS
gradient gel (Biorad, Herculues, CA), transferred to a nitrocellulose membrane, blocked
in 5% milk for 1 hour, and probed for NFATc1nuc using an HA antibody (Sigma, St.
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Louis, MO), 1:1000, or GAPDH (Chemicon, Temecula, CA), 1:1000. The blots were
visualized using an HRP-conjugated secondary antibody and chemiluminescence (NEN
Life Science Products, Boston, MA).
Semi- and Quantitative PCR Studies
Islet RNA was isolated as above, and cDNA was prepared using the RETROscript
kit (Ambion, Houston, TX). cDNA was then subjected to semi-quantitative PCR by
standard methods (primer sequences listed in Supplementary Table 1) or quantitative
PCR using Taqman Gene Expression Assays (Applied Biosystems, Foster City, CA).
Each quantitative reaction was carried out in triplicate, and islets from 3-5 mice of each
genotype were independently tested. Data are normalized to ß-actin. Results are
expressed as the mean +/- S.E.M.
Morphometry and immunohistochemistry
Mice were sacrificed and pancreata were immediately isolated, rinsed in PBS,
fixed for 3-4 hours in 4% PFA, and cryoprotected overnight in 30% sucrose. Tissue was
then imbedded in OCT (TissueTek) and 8 µm frozen sections were obtained by
cryosection (Leica). Anitgen retrieval was performed for all nuclear markers using
sodium citrate (Vector Laboratories, Burlingame, CA). Primary antibodies and dilutions:
Insulin (AbCam, Cambridge, MA), 1:200; Pdx1 (Chemicon, Temecula, CA), 1:500;
Glut2 (Alpha Diagnostic International, San Antonio, TX), 1:500; MafA (Bethyl,
Montgomery, TX), 1:100; NFATc1 (7A6 epitope; Ref. 4), 1:200; NFATc2 (Ref. 5),
1:200; NFATc3 (Santa Cruz Biotechnology, Santa Cruz, CA) 1:100; NFATc4 (Santa
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Cruz Biotechnology, Santa Cruz, CA) 1:50. BrdU lableled ß-cells were detected by
insulin and BrdU co-staining using the Anti-BrdU antibody kit (Amersham Biosciences,
Piscataway, NJ) according to the manufacturer’s protocol. All images were collected
using confocal microscopy (Leica) and are representative of over 50 islets from n>3
mice. Apoptotic ß-cell nuclei were detected using a TdT-mediated dUTP nick end
labeling (TUNEL) kit (Roche, Indianapolis, IN) according to the manufacturer’s
protocol; ß-cells were marked using by insulin immunostaining.
ß-cell mass was assessed by point-counting morphometry. Cross-sectional area of
insulin(+) cells were measured at 100 µm intervals and normalized to total pancreatic
area using Image-Pro Plus analysis software (Media Cybernetics, Silver Spring, MD). ßcell mass is expressed in mg after normalization to total pancreas mass.
X-gal staining of brains was performed as described 5. Briefly, brains were
dissected and 500 µM coronal sections were fixed in 4% paraformaldehyde in 100 mM
PBS and 2 mM MgCl2 for 1 hour. Brains were then washed in PBS + 0.1% Triton X-100
and stained overnight at 37˚C in PBS containing 5 mM potassium ferricyanide, 5 mM
potassium ferrocyanide, 2 mM MgCl2, and 1 mg/ml X-gal. After staining, brain sections
were washed in PBS, and images were collected with a dissecting microscope (Zeiss,
Thornwood, NY). n=3 RIP-Cre; ROSA26R mice.
Chromatin Immunoprecipitation
MIN6 cells (passage 60-70) were grown in DMEM (Invitrogen, Chicago, IL) +
10% FCS (Hyclone, Logan, UT) at 37˚C and 5% CO2. For ChIP studies, MIN6 cells were
transfected with a human NFATc1 expression construct (gift from G. Crabtree) using
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SuperFect Reagent (Qiagen, Valencia, CA). 48 hours after transfection, cells were
stimulated with Phorbol 12-myristate 13-acetate (PMA) and Ionomycin for 1 hour,
washed in cold PBS, and DNA was crosslinked using 1% formaldehyde for 12 minutes at
room temperature. The crosslinking reaction was quenched using 250 mM glycine, and
cells were washed twice in cold PBS. Cells were then lysed and subjected to chromatin
shearing by sonication (Vibra Cell Cup Horn Sonicator, Sonics and Materials, Inc,
Newtown, CT). Lysate was washed and chromatin immunoprecipatations were
performed using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Upstate, Lake
Placid, NY) according to the manufacturer’s protocol with a monoclonal NFATc1
antibody (BD Pharmigen, San Diego, CA). Immunopreciptated DNA was detected by
PCR (primer sequences listed in Supplementary Table 1).
Statistical Methods
All measurements are presented as the mean +/- S.E.M. Significance was determined
using the two-tailed, unpaired Student’s t-Test, and p<0.05 was considered significant.
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