<p> Purinergic Signalling </p><p>Online Resource</p><p>Role of adenosine A2B receptor signaling in contribution of cardiac mesenchymal stem-like</p><p> cells to myocardial scar formation.</p><p>Sergey Ryzhov, Bong Hwan Sung, Qinkun Zhang, Alissa Weaver, Richard J Gumina, Italo</p><p>Biaggioni, Igor Feoktistov </p><p>Divisions of Cardiovascular Medicine (SR, QZ, RJG, IF) and Clinical Pharmacology (IB),</p><p>Departments of Medicine (SR, QZ, RJG, IF), Cancer Biology (BHS, AW) and Pharmacology</p><p>(IB, IF), Vanderbilt University Medical School, Nashville, TN </p><p>Corresponding author: </p><p>Igor Feoktistov, MD, PhD, </p><p>360 PRB, Vanderbilt University, 2220 Pierce Ave, Nashville, TN 37232-6300. </p><p>Phone: 615-936-1732, </p><p>FAX: 615-936-1733, email: [email protected]</p><p>1 4 0</p><p>1 93% 0% 3 0 1 A - C 2 Control P 0 A 1 1</p><p>0 93% 1 7% 0%</p><p>10 1 10 2 10 3 10 4 FITC-A 4 0</p><p>1 99% 0% 3 0 1 A - C TGF 2 P 0 A 1 1</p><p>0 99% 1 1% 0% 5 0</p><p>1 10 1 10 2 10 3 10 4 D FITC-A C Isotype control (αSMA)</p><p>Online Resource Fig. 1 Negative controls for intracellular αSMA staining in cardiac Sca-</p><p>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</p><p>(TGF-β) of 1 ng/ml TGF-β1 for 24 hours. </p><p>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</p><p> a 0 b 0 0 1 A A 0 1 - - 0 C 0 C</p><p>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 -</p><p>H Live/Dead 6</p><p>- 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</p><p>FSC-A FSC-A 0 0 0 0 2 2 A 0 0 A - - C C</p><p>1 2 3 4 S 1 2 3 4</p><p>S 10 10 10 10 10 10 10 10 S F Alexa Fluor 350-A A 450-A Live/Dead Isotype control control</p><p>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</p><p>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</p><p>CD45- cells gate.</p><p>3 4 4 4</p><p>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</p><p>3 1 2 3 4 1 2 3 4 1 2 3 4</p><p>D 10 10 10 10 10 10 10 10 10 10 10 10</p><p>C PE-A PE-A PE-A</p><p>Isotype control (Sca-1)</p><p>Online Resource Fig. 3 Negative controls for Sca-1 cell-surface staining of cardiac CD45- myocyte-free cell populations.</p><p>Representative cytofluorographic dot plots of negative Sca1+ staining with an isotype-matched</p><p>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. </p><p>4 4</p><p> d0 0 1 0.1% 0.1% 3 0 1 A -</p><p>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</p><p> o PE-Cy7-A s I Isotype control (Col1) 4</p><p> d7 0 1 0.1% 0.1% 3 0 1 A -</p><p>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</p><p> o PE-Cy7-A s I Isotype control (Col1) 4 0 d14 1 0.1% 0.0% 3 0 1 A -</p><p>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</p><p> o A 450-A s I Isotype control (Col1)</p><p>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-</p><p>5 stromal cell populations obtained from heart ventricles before (d0) and on days 7 (d7) and 14</p><p>(d14) after MI. </p><p>6</p>