Purinergic Signalling

Online Resource

Role of adenosine A2B receptor signaling in contribution of cardiac mesenchymal stem-like

cells to myocardial scar formation.

Sergey Ryzhov, Bong Hwan Sung, Qinkun Zhang, Alissa Weaver, Richard J Gumina, Italo

Biaggioni, Igor Feoktistov

Divisions of Cardiovascular Medicine (SR, QZ, RJG, IF) and Clinical Pharmacology (IB),

Departments of Medicine (SR, QZ, RJG, IF), Cancer Biology (BHS, AW) and Pharmacology

(IB, IF), Vanderbilt University Medical School, Nashville, TN

Corresponding author:

Igor Feoktistov, MD, PhD,

360 PRB, Vanderbilt University, 2220 Pierce Ave, Nashville, TN 37232-6300.

Phone: 615-936-1732,

FAX: 615-936-1733, email: [email protected]

1 93% 0% 3 0 1 A - C 2 Control P 0 A 1 1

0 93% 1 7% 0%

10 1 10 2 10 3 10 4 FITC-A 4 0

1 99% 0% 3 0 1 A - C TGF 2 P 0 A 1 1

0 99% 1 1% 0% 5 0

1 10 1 10 2 10 3 10 4 D FITC-A C Isotype control (αSMA)

Online Resource Fig. 1 Negative controls for intracellular αSMA staining in cardiac Sca-

1+CD31- cells. Representative cytofluorographic dot plots of negative αSMA staining with an isotype-matched IgG in cardiac Sca-1+CD31- cells cultured in the absence (Control) or presence

(TGF-β) of 1 ng/ml TGF-β1 for 24 hours.

2 0 0 0 0 0 0 1 c e 1 R4 0 0 0 R3 0 8 8 0 0 0 0 A A - - 6 6 C C S S F 0 0 S 0 0 4 4 0 0 0 0 2 0 2 0 0 0

a 0 b 0 0 1 A A 0 1 - - 0 C 0 C

0 1 2 3 4 S 0 10 10 10 10 S 10 1 10 2 10 3 104 8 F 8 R2 S Alexa Fluor 350-A A 450-A 0 0 0 A 0 -

H Live/Dead 6

- CD45 6 C C S S 0 S F 0 0 0 0 0 0 0 4 4 0 R1 0 d 1 f 1 0 R4 0 0 0 0 0 2 0 0 2 R3 8 8 0 0 0 0 0 0 H A A A - - - - 6 6 C C C C 0 200 400 600 800 1000 S 0 200 400 600 800 1000 S F S S 0 0 FSC-A FSC-A S F S 0 0 4 4

FSC-A FSC-A 0 0 0 0 2 2 A 0 0 A - - C C

1 2 3 4 S 1 2 3 4

S 10 10 10 10 10 10 10 10 S F Alexa Fluor 350-A A 450-A Live/Dead Isotype control control

Online Resource Fig. 2 Gating strategy for the analysis of myocyte-depleted single cell suspensions obtained from heart ventricles. a SSC-A/FSC-A profile of cardiac cell suspension and parental gate (R1); b Daughter gate of forward-scatter height versus forward-scatter area was used to eliminate doublets (FSC-H vs

FSC-A plot, R2 gate); c Subsequent gate R3 was used to discriminate between live (Blue fluorescent dye-negative) and dead cells (Blue fluorescent dye-positive); d Cells incubated in the absence of Blue fluorescent reactive dye were used as a negative control for live/dead cell staining; e Analysis of CD45 expression versus SSC-A was used to gate non-immune cell population (R4). f Cells incubated in the presence of isotype-matched IgG were used to setup

CD45- cells gate.

3 4 4 4

0 d7 0 d14 0 d0 1 1 1 3 3 3 0 0 0 1 1 1 A A A - - - C C C 2 2 2 P P P 0 0 0 A A A 1 1 1 1 1 1 0 0 0 1 0.0% 1 0.0% 1 0.0% 1

3 1 2 3 4 1 2 3 4 1 2 3 4

D 10 10 10 10 10 10 10 10 10 10 10 10

C PE-A PE-A PE-A

Isotype control (Sca-1)

Online Resource Fig. 3 Negative controls for Sca-1 cell-surface staining of cardiac CD45- myocyte-free cell populations.

Representative cytofluorographic dot plots of negative Sca1+ staining with an isotype-matched

IgG and anti-CD31 antibody of CD45- myocyte-free cell populations obtained from heart ventricles before (d0) and on days 7 (d7) and 14 (d14) after MI.

d0 0 1 0.1% 0.1% 3 0 1 A -

C 0.1% 2 T I 0 F 1 l o r 1 t ) 0 n 1 o A c M 99.3% 0.5% e S p α ( y 10 1 10 2 10 3 10 4 t

o PE-Cy7-A s I Isotype control (Col1) 4

d7 0 1 0.1% 0.1% 3 0 1 A -

C 0.1% 2 T I 0 F 1 l o r 1 t ) 0 n 1 o A c M 99.7% 0.2% e S p α ( y 10 1 10 2 10 3 10 4 t

o PE-Cy7-A s I Isotype control (Col1) 4 0 d14 1 0.1% 0.0% 3 0 1 A -

C 0.1% 2 T I 0 F 1 l o r 1 t 0 ) n 1 o A c M 99.7% 0.2% e S p α ( y 10 1 10 2 10 3 10 4 t

o A 450-A s I Isotype control (Col1)

Online Resource Fig. 4. Negative controls for intracellular αSMA and collagen I staining of cardiac Sca-1+CD31- stromal cell populations. Representative cytofluorographic dot plots of negative αSMA and collagen I staining with control isotype-matched antibodies of Sca-1+CD31-

5 stromal cell populations obtained from heart ventricles before (d0) and on days 7 (d7) and 14

(d14) after MI.