1. 3΄,4΄-Dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of EGF receptor Eun Jin Jang 1#, Young Mi Seok 2#, Jae In Lee 3, Hyun Min Cho 1, Uy Dong Sohn 4 and In Kyeom Kim 1,2,* 1 Department of Pharmacology, 2 Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea; 3 Department of Chemistry, Duksung Womens University, Seoul 132-714, Korea: 4 College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea. # These authors equally contributed to this paper. *Correspondence and Proofs In Kyeom Kim, M.D., Ph.D. Department of Pharmacology Kyungpook National University School of Medicine 101 Dongin-2-Ga Daegu, 700-422, Republic of Korea Tel: +82-53-420-4833 Fax: +82-53-426-7345 E-mail: inkim@knu.ac.kr Supplementary Figures

2. O Supplementary Fig. 1. Supplement Fig. 1. Chemical structure of 3,4-dimethoxythioflavone.

3. Supplementary Fig. 2. (a) (b) (c) (d)

4. Supplement Fig. 2. Effect of ATP-sensitive K + channels blocker glibenclamide (GB) and nonselective K + channel blocker tetraethylammonium (TEA) on 3΄,4΄- dimethoxythioflavone-induced vasorelaxation. 3΄,4΄-Dimethoxythioflavone was added cumulatively to elicit relaxation when vascular contractions induced by KCl (50 mmol/L; a, c) or U46619 (100 nmol/L; b, d) reached plateaus in endothelium-intact rat aortic rings. Rings were pretreated with GB (1.0 or 10 μmol/L; a, b), TEA (10 or 100 μmol/L; c, d), or vehicle (0.1% DMSO) for 30 min. Relaxation is expressed as a percentage of the maximal contraction. Data are the mean ± SEM of n=4 experiments.

5. (a) (b) Supplementary Fig. 3.

6. Supplement Fig. 3. Effect of cyclooxygenase blockers indomethacin on 3΄,4΄- dimethoxythioflavone-induced vasorelaxation. 3΄,4΄-Dimethoxythioflavone was added cumulatively to elicit relaxation when vascular contraction induced by KCl (50 mmol/L; a), U46619 (100 nmol/L; b) (b) reached plateaus in endothelium-intact rat aortic rings pretreated with indomethacin (1.0 or 10 μmol/L) or vehicle (0.1% DMSO) for 30 min. Relaxation is expressed as a percentage of the maximal contraction. Data are the mean ± SEM of n=4 experiments.

7. Supplementary Fig. 4. (a) (b)

8. Supplement Fig. 4. Effect of muscarinic receptor antagonist atropine on 3΄,4΄- dimethoxythioflavone-induced vasorelaxation. 3΄,4΄-Dimethoxythioflavone was added cumulatively to elicit relaxation when vascular contractions induced by KCl (50 mmol/L; a) or U46619 (100 nmol/L; b) reached plateaus in endothelium-intact rat aortic rings pretreated with atropine (1.0 or 10 μmol/L) or vehicle (0.1% DMSO) for 30 min. Relaxation is expressed as a percentage of the maximal contraction. Data are the mean ± SEM of n=4 experiments.

9. ET (-) (b) (d) (a) ET (-) (c) Supplementary Fig. 5.

10. Supplement Fig. 5. Effect of a Src inhibitor PPT and an ERK inhibitor U0126 on 3΄,4΄-dimethoxythioflavone-induced vasorelaxation. 3΄,4΄- Dimethoxythioflavone was added cumulatively to elicit relaxation when vascular contractions induced by KCl (50 mmol/L; a, c) or U46619 (100 nmol/L; b, d) reached plateaus in endothelium-denuded rat aortic rings pretreated with PPT (0.1, 1.0 or 10 μmol/L), U0126 (0.1, 1.0 or 10 μmol/L), or vehicle (0.1% DMSO) for 30 min. Relaxation is expressed as a percentage of the maximal contraction. Data are the mean ± SEM of n=4 experiments.

11. Constractor KCl (mN)U46619 (mN)Phenylephrine (mN) ET (+) 30.0 ± 3.940.0 ± 6.925.0 ± 5.1 ET (-)35.8 ± 1.843.5 ± 4.736.0 ± 2.3 Table. 1. The maximum tension (mN) produced by KCl, U46619 or phenylephrine in rat aortic rings with intact or denuded-endothelium Data are expressed as means ± SEM of four experiments.