Supplemental
Figures
Supplemental Figure
1
Supplemental
Figure
1.
AdipoChaser mouse model
s used for cold and thermoneutral exposures.
(
A
)
AdipoChaser
-LacZ mouse model, the dox-
based, tet
-responsive, Cre
-loxP labeling system. AdipoChaser
-LacZ
mice
are derived
from interbreeding three transgenic strains: 1) mice expressing the “tet
-on” transcription factor
rtTA under the control of the adipone
ctin gene promoter (Adn-
rtTA); 2) mice expressing tet-
responsive CRE
(TRE
-Cre) that is activated by rtTA in the presence of dox
; and
3) reporter mice expressing a LacZ reporter gene
(encodes β
-galactosidase) from the Rosa26 locus in a Cre
-dependent manner (Rosa26-
loxP
-STOP
-loxP
-LacZ).
AdipoChaser
-LacZ mouse
expresses rtTA in adiponectin expressing cells (
e.g.
all the white adipocytes
), but
does not express LacZ in any cell type while maintained on food not containing dox
. When dox is included
in
the food, c
ells that express rtTA will have the
TRE
promoter activated so that
Cre
expression
is induced
. The
Cre protein will specifically cut out the floxed transcriptional stop cassette and then turn on LacZ expression.
Even after the
withdrawal
of dox from the food, these LacZ positive cells will permanently express LacZ.
(
B
)
10
-week
-old Wild
-type (WT) male mice were housed at 24
o
C, 6
o
C, or 30
o
C for 1 week.
qPCR analysis shows
the mRNA expression levels of
Ucp1
,
Adipoq
,
Ppargc1a,
as well as
Cidea
in BAT of WT mice. n = 5 male mice.
Data represent mean ± s.d. of biologically independent samples.
**P < 0.01. Statistical significance was assessed
using one
-way ANOVA followed by Tukey’s multiple comparisons test.
(
C
) AdipoChaser
-LacZ male mice were
kept on normal chow until 10 weeks of age. Mice were then treated with β3
-
adrenergic receptor agonist for 7
days, during the last 4 days, mice were switched to dox-
containing chow diet. (
D
) Representative X
-gal staining
of BAT from AdipoChaser
-LacZ mi
ce treated with β3
-adrenergic receptor (CL
-316243) agonist. Image is
representative of two independent experiments.
(
E
) Quantification of the percentage of LacZ
-positive brown
adipocytes in the total brown adipocytes. n = 3 mice (saline); 12 mice (CL). Dat
a represent mean ± s.d. of
biologically independent samples, **P < 0.01. Statistical significance was assessed using a Mann Whitney test.
(
F
) Body
weight of 10-
week
-old
AdipoChaser
-LacZ male mice before and after 7
days of temperature switch. n
= 5 mice/gr
oup.
(
G
) BAT weight of
AdipoChaser
-LacZ male mice after 7
days of temperature switch. n = 5
mice/group. These images are representative of three independent experiments.
(
H
) The average
cell size of
LacZ negative and positive brown adipocytes in
AdipoChase
r-LacZ male mice after 7
days of temperature
switch. n = 333 cells (6
o
C LacZ
-); 324 cells (6
o
C LacZ+); 200 cells (24
o
C LacZ
-); 161 cells (24
o
C LacZ+).
Supplemental Figure 2
Supplemental
Figure 2.
Brown adipocytes do not undergo apoptosis even after prolonged cold exposure.
(
A–C
) Cleaved (activated) Caspase 3 (green) and Perilipin (
red
) immunofluorescence staining in BAT of WT
mice kept at 24
o
C or expose to 6
o
C for indicated time. Scale bar: 50
μm. (
D
) Quantification of the percentage of
cleaved Caspase 3+ brown adipocytes in the total brown adipocytes. n = 4 mice (24
o
C); 3 mice (6
o
C 1 week); 5
mice (6
o
C 4 weeks).
All images are representative of three independent experiments.
Data represent mean
± s.d.
of biologically independent samples.
Supplemental Figure 3
Supplemental
Figure
3.
Adiponectin low expressing brown adipocytes have higher mitochondrial
membrane potential
(
A
) AdipoChaser
-mT/mG mouse model, similar to the AdipoChaser
-LacZ mouse, is
produced by crossing
Adn
-
rtTA transgenic mice with
TRE
-Cre and
Rosa26
-loxP
-STOP-
loxP
-mT/mG
transgenic mice.
All cells
in
AdipoChaser
-mT/mG mouse express tdTomato. W
hen dox
is included
in the food,
adiponectin expressing cells will have the
TRE
promoter activated so that
Cre
expression
is induced. The Cre
protein will specifically cut out the floxed tdTomato gene and thus turn on
eGFP
expression.
(
B
) AdipoChaser
-
mT/mG male mice were kept
on normal chow until 10
weeks of age. Mice were then fed
with dox
-containing
chow for 4
days.
(
C
) Representative
immunofluorescence staining for Perilipin (red), GFP (green) and DAPI
(blue) of BAT from AdipoChaser
-mT/mG mice.
Scale bar: 50 μm.
Th
ese images are representative of three
independent experiments. (
D
)
AdipoChaser
-mT/mG male mice were kept on normal chow until 10 weeks of
age. Mice were then treated with dox
-containing chow diet for one week. (
E
)
Representative
immunofluorescence signal from GFP+ (green) or Tomato+ (red) primary brown adipocytes from AdipoChaser-
mT/mG mice kept at 24
o
C, stained with MitoTraker Deep Red (labels mitochondria and reflects mitochondrial
membrane potential) (blue). Scale bar: 50 μm. (
F
) Quantification of MitoTraker Deep Red fluorescent signal in
GFP+ or Tomato+ cells. n = 13 cells for each group. Data represent mean ± s.d. of biologically independent
samples, **P < 0.01. Statistical significance was assessed using a two
-tailed Student’
s t-test (
F
). All images are
representative of three independent experiments.
(
G
)
AdipoChaser
-YFP mouse model, similar to the
AdipoChaser
-LacZ and AdipoChaser
-mT/mG mice, this mouse model is produced by crossing
Adn
-rtTA
transgenic mice with
TRE
-
Cre and
Ro
sa26
-
loxP
-STOP-
loxP
-YFP transgenic mice.
When dox
is included
in
the food, adiponectin expressing cells will have the
TRE
promoter activated so that Cre
expression
is induced
.
The Cre protein will specifically cut out the floxed
transcriptional stop cassette and then turn on YFP
expression.
Supplemental
Figure 4
Supplemental
Figure
4. Single
-cell RNA sequencing identifies differentially expressed genes defining the
brown adipocyte clusters.
Heat map of the
top
20 most differentially
expressed genes that define the primary
brown adipocyte clusters BA
-L, BA
-H1, BA-
H2, BA-
H3, as well as WA and NA depicted in Figure
3A
. This
data is from a single experiment.
Supplemental
Figure 5
Supplemental Figure 5.
Single
-cell RNA sequencing reveals genes enriched in BA
-H cluster: OXPHOS.
(
A
) Distribution of
Ndufa1
,
Ndufa2
,
Ndufa5
, and
Ndufa11
expression within
tSNE
plot, showing genes related
to OXPHOS complex I are enriched
within the BA
-H cluster.
(
B
) Distribution of
Sdha
and
Sdhd
expression
within
tSNE
plot, showing genes related to OXPHOS complex II are enriched
within the BA
-H cluster. (
C
)
Distribution of
Uqcrc1
and
Uqcr11
expression within tSNE
plot, showing genes related to OXPHOS complex
III
are enriched
within the BA
-H cluster. (
D
) Distribution of
Cox5a
,
Cox6b1
,
Cox7a2
, and
Cox8b
expression
within
tSNE
plot, showing genes related to OXPHOS complex IV
are enriched
within the BA
-H cluster.
(
E
)
Distribution of
Atp5a1, Atp5d,
Atp5j2
, and
Atp5k
expression within
tSNE
plot, showing genes related to
OXPHOS complex V are enriched
within the BA
-H cluster.