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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

+--


isotype +++F4/80

CD206

F4/80

CD206

DAPI

merged

CD206+iNOS+DAPI

+--


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


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


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.

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