Supplementary Information Mueller et al:
Migration and Homeostasis of Naïve T cells Depends on Coronin 1-mediated
Pro-Survival Signals and not on Coronin 1-dependent F-Actin Modulation
Supplementary Figures
Figure S1: Phenotypic characterization, thymocytes and T-cells in wild type and
coronin 1 deficient mice.
(A) T-cells from wild type and coronin1 deficient mice were lysed in SDS sample buffer,
separated by SDS page and immune-blotted for coronin 1 and Actin. (B) Single cell
suspensions from thymus, spleen and lymph nodes were stained for CD4 positive cells
and the indicated cell populations quantitated. (C) Annexin V stainings of CD4+ SP
thymocytes from wild type and coronin 1 deficient animals were quantitated. Quantitation
was carried out with data obtained from at least 3 animals. Depicted are mean +/- SD. (D)
Single cell suspensions from thymus, spleen and lymph nodes were stained for CD4 and
CD62L positive cells and the indicated cell populations quantitated. (E) Dot blot of
thymus suspensions from representative wild type and coronin 1 deficient animals stained
with the indicated antibodies. (F) CD4+ single positive thymocytes from wild type and
coronin 1 deficient animals were stained with the indicated antibodies. Experiments were
repeated at least three times. Shown are representative contour blots.
Figure S2: Phalloidin fluorescence, F/G actin ratio and apoptosis in siRNA
transfected Jurkat T-cells and coronin 1 expressing Mel JuSo cells. The human Jurkat
T-cell line was transfected for 5 days with human anti-coronin1 siRNA or a control
siRNA specific for the murine coronin 1 (20). (A) Proteins from cell lysates obtained
from the same cells as used under B-D were separated by SDS-PAGE and immunoblotted
using anti-coronin 1 or anti-actin antibodies. (B) Jurkat T-cells transfected with either the
human or control mouse anti-coronin 1 siRNA were analyzed for their G- and F-actin
content. Cells were lysed and proteins present in supernatants (S) and pellets (P) prepared
as described above were separated by SDS-PAGE and immunoblotted using anti-actin
antibodies. (C) The cells used in B were stained using AlexaFluor 568 phalloidin and
analyzed by FACS. (D) Cells as under B and C were stained with PE-conjugated Annexin
V and analyzed by FACS. Depicted are means +/- SD of cells from three cell samples
analyzed individually. Cells as above were incubated for 0, 4 or 6 hours with 20 ng/ml of
human TNFα to induce apoptosis (D, insert) . (E-H) Mel JuSo cells stably expressing
coronin 1 or coronin 1 negative control cells [1] were analyzed as described for the
siRNA treated Jurkat T-cells under A-D with the exception of panel H were staurosporin
(1 μM) was used instead of TNFα (see insert). Experiments were repeated at least two
times in triplicates.
Figure S3: Staurosporin induced apoptosis of wild type and coronin 1 deficient Bcells
Wild type and coronin 1 deficient B-cells were incubated for the indicated time with
DMSO (carrier) (C) or staurosporin (STS, 1 μM) and the percentage of viable cells was
determined using Annexin V and 7-AAD labeling. Experiments were repeated three
times.
Figure S4: Spontaneous apoptosis and migration of differentially treated naïve
splenic T-cells and single positive thymocytes in the presence and absence of coronin
1
(A/B) Spontaneous apoptosis after 20 hours and transwell migration (3h against 300
ng/ml of CCL19) of naïve splenic T-cells and (C/D) single positive thymocytes were
analyzed after migration (3h against 300 ng/ml of CCL19), incubation in migration
medium (3h) only or migration medium containing 300 ng/ml of CCL19 (3h) as a control.
Depicted are means +/– SD of duplicates. The experiments were performed at least 2
times.
Figure S5: T-cell signaling, proliferation and cellular calcineurin levels in the
presence and absence of coronin 1.
(A) Calcium measurements in Fluo-3 loaded T-cells were performed as described in
methods. The first arrow indicates the addition of antibodies in calcium free medium
whereas the second arrow indicates the addition of calcium. (B) Proliferation of coronin 1
deficient and wild type naive T-cells using the indicated stimuli as described in methods.
Depicted are means +/– SD of triplicate cultures. (C) FACS analysis of Calcineurin Aβ
expression in CD4+ single positive thymocytes or naïve splenic T-cells. (D) Calcineurin
activity was analyzed in naive coronin 1 deficient and wild type T-cells left untreated or
activated with α-CD3/28 and cross linking secondary antibody for 15 minutes, as
described in methods. Depicted are means +/– SD of triplicates. (E) Calcineurin activity
was analyzed in naive coronin 1 deficient and wild type T-cells left untreated or activated
with PMA/Ionomycin for 15 minutes, as described in methods. Depicted are means +/–
SD of triplicates from a representative experiment out of at least three independent ones.
Figure S6: T-cell signaling in the presence and absence of coronin 1.
Mature CD4 SP thymocytes (CD24 low and CD62L high) were labeled with α-CD3 (10
μg/ml) and α-CD28 (5 μg/ml) for 20 min on ice, washed and cross linked with secondary
mouse α-hamster in pre-warmed RPMI for the indicated time at 37°C in a waterbath.
Cells were then washed in ice cold medium, fixed using 4% PFA and processed for
intracellular staining using α-CD3ζ or α-phospho-CD3ζ antibodies (both from BD) or the
corresponding isotype control (Cell Signaling). Experiments were repeated two times in
triplicates using T-cells isolated from different mice.
Supplementary Movie 1 and 2:
Thymocytes from coronin 1 wild type and coronin 1 deficient mice were prepared as
detailed in the supplementary methods and seeded on ICAM-1 coated chambered
coverslips at 37°C. Video microscopy was applied to track the migration of single cells.
The chemo attractants CCL19 was directly added on the top, and images were taken at a
frequency of 6 frames per min using a 40X objective. Experiments were repeated more
than three times.
Figure S1
SP CD4+ Thymus
6
4
2
3
2
F
0
8
6
4
2
8
6
4
2
+/+
22.8
55.4
1
15.9
23.3
83.1
5.23
-/-
1.14
75
19
20.7
58.5
13.7
20.5
CD62L
-/-
5.2
67.1
8.27
CD8
18.2
34.1
+/+
CD69
6.88
58.6
CD24
53.5
1.83
2
8.14
3
0
+/+
-/-
4
0
CD4/62L+ Lymphnode
5
26.4
1
81.1
6.86
2
10
0
3
CD4/62L+ Spleen
10
0
CD4
2
cell numbers (106)
4
1
E
4
SP CD4/62L+ Thymus
D
+/+
-/-
5
6
+/+
-/-
4
0
CD69
% Annexin V
6
5
8
0
cell numbers (106)
C
10
8
+/+ -/-
CD4+ Lymphnode
cell numbers (106)
10
cell numbers (106)
- Coro 1
- Actin
CD4+ Spleen
cell numbers (106)
B
cell numbers (106)
A
CD62L
CD24
Figure S2
Jurkat T cells
(kDa) msiRNA hsiRNA
93 -
msiRNA hsiRNA
52 -
93 -
(kDa) Control Cor 1
93 52 -
Actin
msiRNA
(kDa)
E
-37
Coronin 1
B
-52
S
F
P
S
(kDa)
93 -
P
52 -
100
msiRNA
hsiRNA
G
Control
Mel-Cor1
80
60
40
20
60
40
20
0
0
200
400
600
800 1000
Phalloidin
25
15
% apoptosis
20
10
5
0
0 4 6
0 4 6
time (h)
10
5
0
msiRNA hsiRNA
0
H
A n n e x in V ( % P o s itiv e c e lls )
0
A n n e x in V ( % P o s itiv e c e lls )
Coronin 1
Transfected
S
P
- Actin
100
% of ma x
% of ma x
80
D
Control
S
P
37 -
37 -
C
Actin
52 -
- Actin
- 52
- 37
Coronin 1
hsiRNA
Control Cor 1
-
200 400 600 800 1000
Phalloidin
10
8
6
60
% apoptosis
A
Mel JuSo
40
20
0
0 1 2 3
0 1 2 3
time (h)
4
2
0
Control Cor 1
Figure S4
CD62Lhigh CD44low splenic naive T-cells
A
B
100
90
WT
KO
90
80
80
70
70
60
60
% viable cells
% migrated cells
WT
KO
50
40
50
40
30
30
20
20
0
0
d
d
te
ba
cu
in
epr
te
ra
ig
9
L1
CC
d
d
te
ba
cu
in
epr
te
ra
ig
m
9
L1
CC
l
ro
nt
co
m
10
l
ro
nt
co
10
Single positive thymocytes
C
D
100
90
WT
KO
90
80
80
70
60
60
% viable cells
% migrated cells
70
50
40
50
40
30
30
20
20
10
10
0
d
te
ba
cu
in
epr
d
te
ra
ig
m
9
L1
CC
l
ro
nt
co
d
te
ba
cu
in
epr
d
te
ra
ig
m
9
L1
CC
l
ro
nt
co
0
WT
KO
Figure S5
A
+/+ Control
+/+ anti CD3/28
-/- anti-CD 3/28
90
WT
200
KO
CPM (103)
Relative calcium flux
120
B 250
60
30
150
100
50
0
0
0
100
200
300
400
500
C
CD3
PMA/Iono
Time (sec.)
C
CD4+ SP Thymocytes
100
100
% of Max
% of Max
-/- Control
+/+ Control
-/- anti-CN
+/+ anti-CN
80
80
60
40
60
40
20
20
0
CD4+ naive splenic T-cells
0
10
100
0
1000 10000
0
10
100
1000 10000
Calcineurin Ab
WT
160
KO
140
120
100
80
60
40
20
0
Control
CD3 / CD28
E
PO4 (nmol/mg protein)
PO4 (nmol/mg protein)
D 180
180
160
WT
KO
140
120
100
80
60
40
20
0
Control
PMA/Iono