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Mu et al. Supplemental Information Supplemental Methods: Animals and MCEC cell line Mice were maintained and bred in the Division of Laboratory Animal Resources at the University of Cincinnati Medical Center. All animal experiments conformed to the Guidelines for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences published by the National Institutes of Health. The protocol for animal experiments was approved by the University of Cincinnati Animal Care and Use Committee (Animal Welfare Assurance Number: A3295-01). C57BL/6 wild type mice (20-25 g) were subjected to cecal ligation and puncture (CLP) surgery or intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (15 μg/g). Sham-operated or PBS-injected mice were used as controls. MCECs were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, New York) supplemented with 10 mM penicillin

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Page 1: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

Mu et al. Supplemental Information

Supplemental Methods:

Animals and MCEC cell line

Mice were maintained and bred in the Division of Laboratory Animal Resources at the

University of Cincinnati Medical Center. All animal experiments conformed to the

Guidelines for the Care and Use of Laboratory Animals prepared by the National

Academy of Sciences published by the National Institutes of Health. The protocol for

animal experiments was approved by the University of Cincinnati Animal Care and Use

Committee (Animal Welfare Assurance Number: A3295-01). C57BL/6 wild type mice

(20-25 g) were subjected to cecal ligation and puncture (CLP) surgery or intraperitoneal

injection of Escherichia coli lipopolysaccharide (LPS) (15 μg/g). Sham-operated or PBS-

injected mice were used as controls.

MCECs were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand

Island, New York) supplemented with 10 mM penicillin / streptomycin (Gibco), 10 mM

HEPES (Sigma), and 5% fetal bovine serum (FBS, Gibco).

Isolation of exosomes

Exosomes were isolated with Total Exosome Isolation Reagent (Invitrogen) following

the manufacturer’s instructions. Briefly, frozen serum samples were thawed at room

temperature and centrifuged at 2,000 g for 30 min to remove any cellular debris. The

supernatant containing cell-free serum was transferred to a fresh tube and each sample

was combined with 1/5th volume of the Total Exosome Isolation Reagent. After

Page 2: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

incubation at 4 °C for 30 min, the samples were centrifuged at room temperature at

10,000 g for 10 min. The supernatant was aspirated and discarded. The exosome pellet

was resuspended in PBS buffer (50–300 μl) and stored at 4 °C for a short term (24 h) or

-80 °C for long term.

In vitro endothelial permeability assay

Transendothelial Electrical Resistance (TEER) Measurement:

To measure TEER, the insert with MCEC was transferred to a resistance measurement

chamber (ENDOHM-12; World Precision Instruments, Sarasota, FL, USA). TEER

measurements were performed using an EVOM2 volt-ohmmeter (World Precision

Instruments, Sarasota, FL). At indicated time intervals, the electrical resistance of

individual MCEC monolayer was obtained by subtracting the resistance of a

corresponding naked insert (no cells) from that of the insert on which MCECs were

grown. Data were collected from triplicate inserts per treatment in each of separate

experiments.

Fluorescein isothiocyanate-dextran flux assay:

After treatment, fluorescein isothiocyanate (FITC)-dextran (10 kDa) (Sigma) stock

solution was added to the medium of the upper chamber to achieve a final

concentration of 1 mg/ml. 50 µl medium for each sample was taken in triplicates from

the lower chamber at various time points and placed in a 96-well cluster plate to

measure fluorescent intensity (excitation at 485 nm and emission at 535 nm). In all

cases, the volume of the basal chamber was maintained at 1.5 ml by replacing 50 µl

sample with 50 µl fresh medium.

Page 3: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

Immunofluorescence staining

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS at room

temperature for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 5 minutes (for

ZO-1 staining, no permeabilization was conducted), and blocked with 2% BSA in PBS

for 1 h at room temperature. Primary antibody was diluted in 1% BSA/PBS and

incubated at room temperature for 1 h. The following primary antibodies were used:

rabbit anti-cortactin (1:300; Abcam); rabbit anti-paxillin (1:250; Abcam), and rabbit anti-

ZO-1(1:200, Invitrogen). All samples were subjected to secondary goat anti-rabbit

antibody conjugated to Alexa 488 nm or 594 nm fluorescence (Invitrogen Corporation,

Molecular Probes). As background controls, slides were incubated with the secondary

antibody alone. All images were adjusted to account for non-specific binding of

antibodies. In order to identify podosomes in MCECs, F-actin was co-stained with Alexa

Fluor 488 phalloidin (1:1000; Molecular Probes).

In vivo measurements of cardiac vascular permeability

Evans blue dye (EBD) at 20 mg/kg was intravenously injected with PMA (50 µg/kg),

exosomes (3.6 × 1012 exosomes/kg), or relative vehicles. At 3 h post injection, animals

were euthanized with pentobarbital 300 mg/kg through intraperitoneal injection (I.P.).

Chest was opened and 30 ml of PBS was flushed through the left ventricle. Heart was

removed and frozen. Frozen sections (7-μm in thickness) were observed under a

confocal microscope LSM 710 (Carl Zeiss Microimaging, Jena, Germany). Vascular

leakage corresponding to the amount of dye in the extravascular compartment was

Page 4: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

quantified with Image J software (Wayne Rasband, National Institutes of Health,

Bethesda, MD).

Cardiac function was assessed in vivo at 6 h post injection using transthoracic

echocardiography (iE33 Ultrasound System, Phillips) with a 40-MHz. After the induction

of general anesthesia with isoflurane gas, hearts were imaged in 2-D and M-mode

recorded through the anterior and posterior LV walls. Anterior and posterior wall

thicknesses (end-diastolic and end-systolic) and LV internal dimensions were measured

using a modification of the American Society for Echocardiography leading edge

method from at least three consecutive cardiac cycles on the M-mode tracings. LV

ejection fraction (EF) was calculated as: EF (%) = [left ventricular end-diastolic

dimension (LVEDd)3 - left ventricular end systolic dimension (LVESd)3/(LVEDd)3] × 100.

LV fractional shortening (FS) was determined as [(LVEDd–LVESd)/LVEDd] × 100.

Page 5: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

Supplemental Figure:

Figure S1.

Figure S1. PMA promotes the generation of podosome clusters. MCECs were

seeded on 100 mg/mL collagen-coated coverslips for 12 hours and then treated with

PBS vehicle, PMA (80 ng/ml) or thrombin (5 U/ml) for 0.5 h. After that, cells were fixed

and stained for F-actin (Green), Paxillin (Red), and nuclear (Blue, DAPI). Images were

captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White

arrows represent podosome clusters at the cell periphery. Scale bar: 20 µm.

Paxillin F-actin DAPI Merge

Con

trol

PMA

80

ng/m

l

Page 6: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

Figure S2.

Figure S2: The sizes of non-septic and septic exosomes were determined by

nanoparticle tracking analysis. Exosomes were isolated with Total Exosome Isolation

Reagent (Invitrogen). The size of exosomes was determined using a NanoSight NS300

(Malvern Instruments Ltd).

Page 7: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

Figure S3.

Figure S3: The viability of MCEC was analyzed after septic exosome and non-

septic exosome stimulation. At 2 h post exosome or PMA exposure, the cell culture

media were collected, and LDH activities were measured. MCECs incubated with 2 %

Triton X-100 were used as control. Values are presented as means ± SD. NS: not

significant (n = 6, p > 0.1).

Supplementary Video 1: Real-time imaging of MCECs.

MCECs transfected with Cortactin-pmCherryC1 (red) and mEGFP-Lifeact-7 (green)

were seeded on 35 mm glass bottom dish and imaged in real time with a TIRF

microscope for 5 min using a 100x objective. Pictures were taken every 10s. Scale bar:

10 µm.

Supplementary Video 2: Real-time imaging the formation of podosome clusters in

MCECs during PMA treatment.

0

50

100

150

Cyt

otox

icity

(%)

LDH Assay

NS NS NS

Page 8: Lippincott Williams & Wilkins · Web viewImages were captured with confocal LSM 710 (Carl Zeiss Microimaging, Jena, Germany). White arrows represent podosome clusters at the cell

Cortactin-pmCherryC1 (red) and mEGFP-Lifeact-7 (green) expressing MCECs were

seeded on 35 mm glass bottom dish and then treated with basal medium plus PMA (80

ng/ml) for 20 min. MCECs were imaged in real time with a TIRF microscope for 10 min

using a 100x objective. Pictures were taken every 10s. Red arrows indicate podosome

clusters in PMA-treated MCEC. Scale bar: 10 µm.

Supplementary Video 3: Real-time imaging the formation of podosome clusters in

MCECs during septic exosome treatment.

Cortactin-pmCherryC1 (red) and mEGFP-Lifeact-7 (green) expressing MCECs were

seeded on 35 mm glass bottom dish and then treated with basal medium plus septic

exosomes (1.2 × 1010 exosomes/ml) for 1 h. MCECs were imaged in real time with a

TIRF microscope for 6 min using a 100x objective. Pictures were taken every 10s. Red

arrows indicate podosome clusters in septic exosome treated MCEC. Scale bar: 10 µm.