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1 Supplemental Material for TXI TELNGIECTSI MUTTED (TM)-MEDITED DN DMGE RESPONSE IN OXIDTIVE STRESS-INDUCED VSCULR ENDOTHELIL CELL SENESCENCE Hong Zhan 1, Toru Suzuki 1,2, Kenichi izawa 1, Kiyoshi Miyagawa, and Ryozo Nagai 3 From the Departments of Cardiovascular Medicine, Ubiquitous Preventive Medicine, and Radiation iology, Graduate School of Medicine, The University of Tokyo, Hongo, unkyo, Tokyo , Japan 1 These authors contributed equally to this work Running head: TM mediates endothelial cell senescence 2 To whom correspondence may be addressed: Toru Suzuki, Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Hongo, unkyo, Tokyo , Japan. Tel.: ; Fax: ; torusuzu-tky@umin.ac.jp 3 To whom correspondence may be addressed: Ryozo Nagai, Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Hongo, unkyo, Tokyo , Japan. Tel.: ; Fax: ; nagai-tky@umin.ac.jp 1

2 Supplemental Experimental Procedures Small interference RN () constructs were obtained as Silencer Select Validated from pplied iosystems/mbion (C, US); kt (sense: 5 -GCGUGCCUGCGGUUtt-3 ; antisense: 5 -CUCGUUCUGGUCCGCgg-3 ); p53 (sense: 5 -GUUCUCUGGGCGGtt-3 ; antisense: 5 -UUCCGUCCCGUGUUCca-3 ); CIP1 (sense: 5 -CGGGUCGCUUUUtt -3 ; antisense: 5 -UUGUCUGCUCCUUGtt-3 ); and negative control #1. ntibodies Normal mouse and rabbit IgG antibodies were purchased from Santa Cruz iotechnology (C, US). Supplemental Figure Legends Supplemental Fig. 1. Four higher magnification photographs of representative cells shown in Fig. 1D, 1E, 1F and 1G to better show nuclear foci.,, C & D. Immunofluorescence analysis for () 53P1 (green), () phosphorylated TM (S1981) (red), (C) total TM (red) and (D) (red) in etoposide- or treated HUVECs was used as nuclear stain (blue). Scale bar, 5 μm. Supplemental Fig. 2. Higher magnification photographs of representative cells in Fig. 3 to better show nuclear foci (cells were treated with 1 μm H 2 O 2 for 3 min in the absence or presence of NC or KU-55933). Immunofluorescence analysis of the effects of antioxidant (NC) and TM inhibitor (KU55933) on phosphorylated TM (red) was used as nuclear stain (blue). Scale bar, 5 μm. Supplemental Fig. 3. Immunofluorescence analysis for 53P1 and γ-h2x-s139 in treated HUVECs was used as nuclear stain (blue). Expression of 53P1 and γ-h2x-s139 labeled by green fluorescence in HUVECs was significantly increased with nuclear 2

3 foci formation after H 2 O 2 treatment. Scale bar, 5 μm. Supplemental Fig. 4. Effects of silencing (RNi) of signaling molecules in the kt/p53/ pathway.. Western blot analysis of the effects of against kt on expression of total kt, p53 phosphorylation (S15) and induction.. Western blot analysis of the effects of against p53 on expression of total p53 and induction. C. Western blot analysis of the effects of against on expression of induction. 1.5x1 5 cells/well were transfected with against kt, p53 and for 72h followed by incubation with 1 μm H 2 O 2 for 3 min in (, ) or 3h in (C). GPDH was used as loading control. Values are mean ± s.e.m (n=3). P<.5 versus cells transfected with the same concentration of negative control. Representative blots are shown in the left panel while corresponding quantitation is shown in the right panels. Reagent only: cells transfected with Lipofectamine 2 alone. Supplemental Fig. 5. Effects of abrogation of gene expression by against kt, p53 or in oxidative stress-induced endothelial senescence.. Staining of S-β-gal activity in cells silenced for kt, p53 or.. Quantitation of percentage of S-β-gal-positive cells in cells silenced for kt, p53 or. Values are mean ± s.e.m (n=3). P<.5 versus cells transfected with the same concentration of negative control (lanes 14, 15 or lane 16, respectively) (n=3 each). Original magnification, 1. Scale bar, 2 μm. Reagent only: cells transfected with Lipofectamine 2 alone. Silencing of kt, p53 or by suppressed increase in S-β-gal-positive cells induced by H 2 O 2. Supplemental Fig. 6. Immunostaining for von Willebrand factor, or p16 in the thoracic aortas of STZ-diabetic TM knockout mice. Six respective TM/ (Wild), TM/- (Hetero), TM-/- (Homo) mice were used.. Immunostaining for von Willebrand factor, an endothelial cell marker, in the thoracic aortas (brown). Normal rabbit IgG antibody was used as negative control. rrows indicate positive staining in the endothelium.. Immunostaining for and p16 (brown). Normal mouse IgG antibody was used as negative control. Scale bar, 5 μm, respectively. Supplemental Fig. 7.,. Western blot analysis of TM expression in thoracic aortas () and kidneys () of TM knockout mice (wild-type, and heterozygous- and homozygous-knockout mice, n=3, respectively). 3

4 Supplemental Fig.1 Etopside - 53P1 H 2 O 2 - TM-S1981 C TM D H 2 O 2-4

5 Supplemental Fig. 2 H 2 O 2 - NC (mm) TM-S1981 TM-S1981 KU (μm) TM-S1981 TM-S1981 5

6 Supplemental Fig. 3 53P1 -H2X-S139 6

7 Supplemental Fig. 4 kt p53-s15 C GPDH kt (pmol) p53 GPDH 1 3 p (pmol) kt/ GPDH(%) p53-s15/ GPDH (%) / GPDH (%) / GPDH (%) kt GPDH (pmol) p53-s15/ GPDH (%) / GPDH (%) (pmol) p (pmol) (pmol) 7

8 Supplemental Fig. 5 H 2 O (Regent only) H 2 O 2 (2 μm) 1 kt 5 1 (pmol) p (pmol) (pmol) (pmol) S- -gal positive cells (%) H 2 O kt p (pmol) 8

9 Supplemental Fig. 6 STZ - Wild Wild Hetero Homo 1 2 L 3 4 (5 μm) normal rabbit IgG antibody 5 6 L 7 8 vwf antibody STZ - Wild 1 2 L Wild 3 Hetero 4 Homo (5 μm) 5 6 L normal mouse IgG antibody 7 8 antibody 9 1 L p16 antibody 9

10 Supplemental Fig. 7 Wild Hetero Homo TM GPDH Thoracic aorta Wild Hetero Homo TM GPDH Kidney 1