Supplementary Digital Content 1 final - Lippincott...
Transcript of Supplementary Digital Content 1 final - Lippincott...
Supplemental Digital Content 1
Supplementary methods
Phagocytosis assay
Mice were intraperitoneally administered with 3 ml of 4% thioglycollate
(Sigma-Aldrich, St Louis, MO, USA). After 3 days, peritoneal macrophages were
collected by peritoneal lavage with 10 ml cold phosphate buffered saline (PBS). Cells
were incubated overnight with Dulbecco’s modified Eagle’s medium at 37°C and 5%
CO2. Non-adherent cells were removed with PBS by repeated washing. Cells were
plated at 1 × 105 cells/well in 96-well flat-bottom plate and incubated with 10 ng/mL
lipopolysaccharide (LPS; Sigma) and 50 µM Rosi, 10nM RvD1, 50 mM DMSO, 64.3
µM ethanol or saline. Phagocytosis of zymosan was assessed by the CytoSelect 96-well
phagocytosis assay according to manufacturer’s instruction (Cell Biolabs, San Diego,
CA, USA). Briefly, 10 µl of zymosan suspension was added to the medium, and then
phagocytosis of zymosan was stopped by the removal of supernatant. Phagocytosis was
evaluated by Infinite 200 and i-control™ – Microplate Reader Software by colorimetric
assay at an absorbance of 405 nm (TECAN, Kawasaki, Japan). Each experiment was
performed in triplicate.
0
1.0
2.0
Alox5siRNA
Alox5
n= 6 6
β-actin
controlsiRNA
control siRNA
Alox5 siRNA
0.5
1.5
*
Intensity(Alox5/β-actin)
Supplementary fig. 1. Knockdown of Alox5 by siRNA reduced the expression of Alox5. Each column represents the mean ± SD. Unpaired Student’s t-test between groups. *P < 0.05. Alox5; arachidonate 5-lipoxygenase.
F4/80
F4/80
iNOS
iNOS
DAPI
+
F4/80+iNOS+DAPI
--
isotypeisotypeisotype
isotype
merged
+++
F4/80+CD206+DAPI
merged
+--
isotypeisotypeisotype
isotype +++F4/80
CD206
F4/80
CD206
DAPI
merged
CD206+iNOS+DAPI
+--
isotypeisotypeisotype
isotype +++CD206
iNOS
CD206
iNOS
DAPI
merged
TUNEL+CD68+DAPI
+--
isotypenegativeisotype
negative +++TUNEL
CD68
TUNEL
CD68
DAPI
merged
Gr-1+DAPI- isotype +Gr-1
Gr-1
DAPI
Supplementary fig. 2. Isotype control to primary antibodies for Gr-1, CD68, F4/80, iNOS, and CD206 were used to evaluate nonspecific background of immunohistochemistry. For negative control of TUNEL assay, control incubation buffer without rTdT enzyme was used. Scale bar, 100 μm.
vehiclevehicle Rosi Rosi
day 2m/m Intact
db Intact
m/m db
CD206
iNOS
DAPI
CD206
iNOS
DAPI
RvD1Rosi+RvD1 RvD1Rosi+RvD1
vehiclevehicle
merged
merged
Rosi Rosi
day 7m/m db
RvD1Rosi+RvD1 RvD1Rosi+RvD1
0
m/m intact
m/m vehiclem/m Rosim/m RvD1m/m Rosi + RvD1
db intact
db vehicledb Rosidb RvD1db Rosi + RvD1
0
0
iNOS+CD206+DAPI+cells/mm2
2 7
CD206+DAPI+cells/mm2iNOS+DAPI+cells/mm2
(day) 2 7 (day)
2 7 (day)
1500
1000
500
1000
800
600
400
200
150
100
50
iNO
S+ C
D20
6+ DA
PI+ c
ells
cel
ls/ m
m2
iNO
S+ D
AP
I+ cel
ls c
ells
/ mm
2
CD
206+ D
AP
I+ cel
ls c
ells
/ mm
2
******
***
***
****
*********
****** *** **
* ********
**
******
******
Supplementary fig. 3. Rosiglitazone coadministered with RvD1 significantly increased
phagocytotic activity of db/db-derived macrophages. Two-way ANOVA (interaction
factor: F6,140 = 10.46, P < 0.0001) followed by separate one-way ANOVA with Tukey
post hoc test for comparison at each time (m/m : F6,70 = 62.87, P < 0.0001; db: F6,70 =
29.17, P < 0.0001). The results are presented as mean ± SD. **** P < 0.0001. LPS;
lipopolysaccharide, DMSO; Dimethly sulfoxide, Rosi; Rosiglitazone. RvD1; Resolvin D1.
3
2
1
0m/m
Opi
tical
Den
sitiy
405
nm
db
+ ++ +
++
++
+++
+ +
+ ++ + + +
- -- - -- - - -
- --
-- - - -- - - - -
- + ++ +
++
++
+++
+ +
+ ++ + + +
- -- - -- - - -
- --
-- - - -- - - - -
-zymosanLPSethanolDMSORosiRvD1
(n=11)
********
****
********
****
****
Supplementary fig. 4. The number of iNOS+CD206+ macrophages did not differ significantly between the groups. Two-way ANOVA (iNOS+CD206+DAPI+: interaction factor: F7,107 = 1.210, P = 0.3037; iNOS+DAPI+: interaction factor: F7,107 = 1.689, P = 0.1192; CD206+DAPI+: interaction factor: F7,107 = 3.862, P = 0.0009) followed by separate one-way ANOVA with Tukey post hoc test for comparing the mean number of CD206+DAPI+ cells at each time (day 2 : F7,55 = 7.632, P < 0.0001; day 7: F7,52 = 9.250, P < 0.0001). The results are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Rosi; Rosiglitazone. RvD1; Resolvin D1. Green, CD206; red, iNOS, and blue; DAPI. Scale bar, 100 μm.
A
B
day 2
DAPI
Gr-1
Gr-1
day 7
merged
salinesaline saline saline DMSO+0.1% ethanol
DMSO+0.1% ethanol
DMSO+0.1% ethanol
DMSO+0.1% ethanol
m/m db m/m db
2 7 (day)
2500
2000
1500
1000
500
0
m/m NS(n=6) (n=6) (n=6) (n=6)
m/m DMSO + 0.1%ethanol db NS db DMSO + 0.1%ethanol
**
**
***
*
Gr-
1+ DA
PI+
cells
/ mm
2
Supplementary fig. 5. Local infiltration of Gr-1+ PMNs was not changed by DMSO coadministered with 0.1% ethanol in both m/m and db/db mice. (A) Infiltration of Gr-1+ PMNs on days 2 and 7 after incision. (B) Total number of Gr-1+ PMNs in subcutaneous tissue was counted. Two-way ANOVA (interaction factor: F3,40 = 3.871, P = 0.016) followed by separate one-way ANOVA with Tukey post hoc test for comparison at each time (day 2 : F3,20 = 0.2858, P = 0.8351; day 7: F3,20 = 15.02, P < 0.0001). The results are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. DMSO; Dimethly sulfoxide. Scale bar, 100µm.
2 72 7
m/m
CD68
DAPI
TUNEL
TUNEL CD68
db
merged
day2
0
1000
800
600
400
200
300
200
100
0(day) (day)
A
C D
DMSO +0.1% ethanolsaline DMSO +
0.1% ethanolsaline
m/m dbday7
DMSO +0.1% ethanolsaline DMSO +
0.1% ethanolsaline
m/m saline m/m DMSO + 0.1% ethanol db saline db DMSO + 0.1% ethanol
2
5 5 6 6 65 5
7
CD68/TUNEL
n = 50
80
60
40
20
(day)
B
CD
68+ TU
NE
L+ DA
PI+
cells
/ mm
2
CD
68+ D
AP
I+ ce
lls/ m
m2
TU
NE
L+ DA
PI+
cells
/ mm
2
Supplementary fig. 6. Phagocytosis of apoptotic cells was not affected by injection
of DMSO and 0.1% ethanol at the incision site. (A) TUNEL+CD68+, TUNEL+DAPI+,
and CD68+DAPI+ cells at the incised sites. (B) The number of CD68+TUNEL+DAPI+
per area at the incision sites. Two-way ANOVA (interaction factor: F3,35 = 2.179,
P = 0.1079). (C) The number of TUNEL+DAPI+ cells per area at the incision sites.
Two-way ANOVA (interaction factor: F3,35 = 1.221, P = 0.3167). (D) The number of
CD68+DAPI+ cells per area at the incision sites. Two-way ANOVA (interaction factor:
F3,35 = 0.4546, P = 0.7157). The results are presented as mean ± SD. DMSO; Dimethly
sulfoxide. Green, TUNEL; red, CD68; blue, DAPI. Scale bar, 100µm.
m/m
iNOS
DAPI
F4/80
db
merged
CD206
DAPI
F4/80
merged
day2ADMSO +0.1% ethanolsaline DMSO +
0.1% ethanolsaline
m/m dbday7
DMSO +0.1% ethanolsaline DMSO +
0.1% ethanolsaline
m/m saline m/m DMSO +0.1% ethanol db saline db DMSO + 0.1% ethanol
400
200
600
0
2 7 (day)
800
500
1500
02 7 (day)
2000 F4/80
500
1500
02 7 (day)
2000 F4/80
100
200
300
02 7 (day)
500
400
CD206
1000
1000
B
C
F4/
80+ iN
OS
+ ce
lls/ m
m2
F4/
80+ C
D20
6+ ce
lls/ m
m2
Tota
l F4/
80+ ce
lls/ m
m2
Tota
l F4/
80+ ce
lls/ m
m2
iNOS
5 5 6 6 65 5n=5
Supplementary fig. 7. Phenotype shift of macrophages from M1 to M2 type was not
affected by injections of DMSO and 0.1% ethanol at the incision site. (A) Infiltration of
F4/80+iNOS+ M1 macrophages and F4/80+CD206+ M2 macrophages was evaluated
on days 2 and 7. (B) The number of F4/80+iNOS+ and total F4/80 macrophages per
area. Two-way ANOVA (F4/80+iNOS+: interaction factor: F3,35 = 3.744, P = 0.0197;
total F4/80+: interaction factor: F3,35 = 1.852, P= 0.1557) followed by separate
one-way ANOVA with Tukey post hoc test for comparison of the number of F4/80+iNOS+
macrophages at each time (day 2 : F3,17 = 4.655, P = 0.0149; day 7: F3,18 = 1.848,
P= 0.1746). (C) The number of F4/80+CD206+ and total F4/80+ macrophages per area.
Two-way ANOVA (F4/80+CD206+: interaction factor: F3,35 = 1.830, P = 0.1598; total
F4/80+: interaction factor: F3,35 = 0.5955, P = 0.6222). The results are presented as
mean ± SD. DMSO; Dimethly sulfoxide. Green, F4/80; red, iNOS or CD206; blue, DAPI.
Scale bar, 100 µm.
Rel
ativ
e ex
pres
sion
(Tgf
b1/ G
3PD
H)
7 (day)
0
2
6
12 12 1212
(day)7
m/m db
m/m db
0
10
5
15
25
n=
n=
11 10 1010
20
4
Tgfb1
Rel
ativ
e ex
pres
sion
(Tnf
/ G
3PD
H)
Tnf
saline
DMSO + 0.1% ethanol
Supplementary fig. 8. Injections of DMSO and 0.1% ethanol did not change gene expression of TNF-α (Tnf) or TGF-β1 (Tgfb1) at the incision site on day 7. Gene expression was quantified by real-time PCR. Two-way ANOVA (tnf: interaction factor: F1,37 = 0.2238, P = 0.6389; Tgfb1: interaction factor: F1,44 = 0.5068, P = 0.4803). Each column represents the mean ± SD. DMSO; Dimethly sulfoxide.