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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 / 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
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.
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
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.
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
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).
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
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.