Supplemental Data
Cyclin-dependent kinase inhibitor, p21 WAF1/CIP1, is involved in adipocyte
differentiation and hypertrophy, linking apoptosis to obesity and insulin resistance
Noriyuki Inoue, Naoya Yahagi, Takashi Yamamoto, Mayumi Ishikawa, Kazuhisa Watanabe,
Takashi Matsuzaka, Yoshimi Nakagawa, Yoshinori Takeuchi, Kazuto Kobayashi, Akimitsu
Takahashi, Hiroaki Suzuki, Hirohito Sone, Alyssa H Hasty, Hideo Toyoshima, Nobuhiro
Yamada, and Hitoshi Shimano.

Supplemental Method
Cell culture and differentiation
Primary MEF cells were harvested from 13.5-day post-coitus embryos of p21(+/+) and p21(-/-) mice.
MEFs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (GIBCO/BRL) with 10%
FCS in 5% CO2.

MEFs were split into 60 mm dishes and cultured in !-modified Eagle’s medium(GIBCO/BRL) with 10% FCS to confluence (Day -2).

At Day 0, medium was replaced with thedifferentiation induction medium A! (5 µg/ml insulin, 1 µM dexamethasone and 0.5 mM IBMX).
This medium was renewed every other day (19).

Real-Time Quantity PCR
2.5µg of total RNA sample was reverse transcribed using High Capacity cDNA Archive Kit(Applied Biosystems).

Expression of each gene was determined by real-time PCR on an ABI

7900HT Sequence Detection System (Applied Biosystems) according to the manufacturer’s
instructions.

Real-time PCR reagents are from Applied Biosystems.

Real-Time Quantity PCR Taq Man probe list
TaqMan Gene Expression Assays (Applied Biosystems)
Gene nameNo.

p21(CDKN1a)

Mm00432448_m1

LeptinMm00434759_m1

Adiponectin

Mm00456425_m1

Resistin

Mm00445641_m1

AdipsinMm00442664_m1

TNF!

Mm00443258_m1

MCP-1

Mm00441242_m1

36B4(acidic ribosomal phosphoprotein P0)

Mm00725448_s1

Figure. A1 Proliferation and adipocyte differentiation of p21(+/+) and p21(-/-) MEFs.

MEF cells were prepared from p21(+/+) and p21(-/-) mice, incubated for 4 days in regular media,
followed by further culture in the adipocyte induction medium for 14 days.
A)

Cell numbers of p21(+/+) MEF (white column) and p21(-/-) MEF(black column) at Day -4(harvest) and Day 0 in a confluent condition just before addition of induction medium.
Dots in the graph are individual cell counts.

Values are means ± SEM.

[p21(+/+) MEFs :n=5 and p21(-/-) MEFs : n=9]""; signicance versus p21 (+/+) MEFs at p<0.01
B)

Proliferation of MEFs determined with Hoechst stain and BrdU uptake at Day 2 of adipocyte
differentiation.

C)

Oil Red O staining of cells at Day 14 of adipocyte differentiation.

Figure. A2

Metabolic parameters in p21 (+/+) and p21 (-/-) mice on a normal chow diet.

p21(+/+) and p21(-/-) mice were maintained on chow diet until 18 weeks of age.
A) Body weight change in p21 (+/+) and p21 (-/-) mice (n=17).
B) Total body mass and lean mass, % fat in p21 (+/+), p21(+/-) and p21 (-/-) mice at 10 weeks ofage as estimated by DEXA (n =18).
C) Weights of epididymal fat pads from p21 (+/+) and p21 (-/-) mice. (n = 14).
D) Glucose tolerance in p21 (+/+) and p21 (-/-) mice.
mg/kg D-glucose.

Mice were intraperitoneally injected with 2Blood glucose (upper panel) and plasma insulin (lower panel)concentrations were measured at the indicated times (n = 11).
E) Insulin tolerance in p21 (+/+) and p21 (-/-) mice.

Mice were injected with 0.5 U/kg insulin.

Blood glucose levels were measured at the indicated times.
Columns and circles; white: p21(+/+), gray: p21(+/-), black: p21(-/-); n=11.
Values are means ± SEM** at p<0.01 for p21(-/-) compared to p21(+/+) groups.

Figure. A3!
diet.

Analysis of white adipose tissue in p21 (+/+) and p21 (-/-) mice on a normal chow

p21(+/+) and p21(-/-) mice were maintained on chow diet until 18 weeks of age.
Size distribution (A) and total cell number (B) of adipocytes from epididymal fat pads.
distribution was as quantified by Coulter counter.

Cell size

Microscopic examination of epididymal white

fat pad (C) and brown adipose tissue (D) (HE staining).
Columns and circles; white: p21(+/+), black: p21(-/-).Values are means ± SEM of 6 mice per group.

Figure. A4!

BrdU uptake in 3T3-L1 adipocytes infected with p21 RNAi adenoviruses

Under the same condition as Figure.1C-E, 3T3-L1 fibroblasts infected with p21 RNAi
adenoviruses at Day -3.! The cells started differentiation at Day 0 with differentiation induction
medium A and changed to differentiation induction medium B at Day 2 (See theExperimental Procedures).

Proliferation of the cells at the indicated days was determined

as estimated by BrdU uptake and Hoechst stain.
A)

Time course analysis for BrdU uptake of 3T3-L1 adipocytes after p21 knockdown at Day -3
Hoechst 33342 for nuclear staining (upper) and BrdU staining (lower) are shown atmagnification of x400.

B)

Quantification of BrdU positive cells (green) ratio corrected to the Hoechst33342 (blue).

Dots in the graph are individual cell counts.
800-1000 cells per group.
cells, * at p<0.05

Values represent mean " SEM from 8-10 fields of*** at p<0.0001 for 137i and 275i versus LacZi adenoviruses infectedFigure.A5

RT-PCR quantification of in white adipose tissue of p21(-/-) mice on a HFHS diet.

mRNA levels of indicated genes were measured by Real Time quantity PCR for RNA samples usedfor northern blot analysis in the Figure.4A.

Expression of each gene was normalized to 36B4

(acidic ribosomal phosphoprotein P0).
** at p<0.01 for p21(+/-) and p21(-/-) versus p21(+/+), * at p<0.05
Columns and circles; white: p21(+/+), gray: p21(+/-), black: p21(-/-); n=6
Values are means ± SEM.

Table. A1

Metabolic parameters of p21 (+/+) and (-/-) mice on a normal diet (18 week old)
FastedFed

p21(+/+)

p21(-/-)

p21(+/+)

p21(-/-)TG (mg/dL)

175 ± 9183 ± 18

118 ± 25

107 ± 14

T-Cho (mg/dL)

103 ± 699 ± 494 ± 985 ± 4

NEFA (mEq/L)

1.43 ± 0.16

1.38 ± 0.08

0.83 ± 0.14

0.64 ± 0.08

Leptin (ng/mL)

0.14 ± 0.09

0.06 ± 0.02

1.05 ± 0.21

0.48 ± 0.07

Values are means ± SEM.

Table. A2(n=16)"; signicance versus p21(+/+) mice at p<0.05 and "" at p<0.01

Metabolic parameters of p21 (+/+) and (-/-) mice on a HFHS dietp21(+/+) and p21(-/-) mice at 10 weeks of age were put fed on a HFHS diet for 6 weeks.
FastedFedp21(+/+)

p21(-/-)

p21(+/+)

p21(-/-)

Glucose (mg/dL)

210 ± 9193 ± 10201 ± 9204 ± 5Insulin (pg/mL)

1727 ± 303

855 ± 185""

5497 ± 994

2625 ± 619"

TG (mg/dL)

93 ± 797 ± 11112 ± 7102 ± 10

T-Cho (mg/dL)

215 ± 6183 ± 13"218 ± 10185 ± 13"

NEFA (mEq/L)

0.68 ± 0.05

0.81 ± 0.07

0.49 ± 0.04

0.56 ± 0.04

Liver TG (mg/g)

35 ± 538 ± 4

N/D

N/D

Liver T-Cho (mg/g)

3.8 ± 0.7

4.7 ± 0.7

N/D

N/D

Leptin (ng/mL)

3.2 ± 1.3

2.1 ± 1.4

8.8 ± 2.7

4.7 ± 1.8

Values are means ± SEM.

(n=6-16)"; signicance versus p21 (+/+) mice at p<0.05 and "" at p<0.01
N/D = not determinedTable. A3

Insulin secretion of p21 knockout mice at normal dietp21(+/+) and p21(-/-) mice were maintained on chow diet.!

Mice were either fasted for 24 h orrefed at 12 h after a 24 h starvation.
FastedRefed

p21(+/+)

p21(-/-)

p21(+/+)

p21(-/-)

Glucose (ng/dL)

81 ± 5.3

81 ± 4.8

201 ± 21208 ± 14

Insulin (pg/mL)

420 ± 190

198 ± 353316 ± 551

5786 ± 2316

Values are means ± SEM.

(n=6)"; signicance versus p21(+/+) mice at p<0.05 and "" at p<0.01