P2P6P10 Hcy Ctrl P2P6 P10 Ctrl Hcy Supplement Fig. I A B Suppl. Fig. I. Cell cycle and apoptotic effects of Hcy treated HUVECs senescence. Primary cultured.

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P2P6P10 Hcy Ctrl P2P6 P10 Ctrl Hcy Supplement Fig. I A B Suppl. Fig. I. Cell cycle and apoptotic effects of Hcy treated HUVECs senescence. Primary cultured HUVECs (P2-P10) were treated with or without Hcy, 25 μM. (A) Cell cycle was detected by flow cytometry and the percentage of cells in the S phase was calculated; apoptosis was detected by TUNEL assay and the percentages of TUNEL-positive cells are expressed. Data was as means±SD (n=4). *P<0.05 vs Ctrl; # P<0.05, vs the Ctrl in their corresponding passages P2P6P10 Ctrl Hcy S phase cells (%) P2P6P10 80 Apoptosis celsl (%) Ctrl Hcy * * * # # # # * * *

Supplement Fig. II Ctrl FA LAR Hcy FA+Hcy LAR+Hcy iNOS mRNA expression A Ctrl FA LAR Hcy FA+Hcy LAR+Hcy 2.5 eNOS mRNA expression B # # ** ## * Suppl. Fig. II. Effect of Hcy on eNOS and iNOS expression. (A) qRT-PCR of eNOS (B) and iNOS (C) mRNA expression in HUVECs (P4-P6) treated with Hcy, 50 μM, with or without pre- supplementation with FA (100 μM) or SAM (100 μM) for 1 hr. *P<0.05, **P<0.01vs. P2 control; # P<0.05 vs. Hcy. Data are mean±SD (n=4).

Supplement Fig. III CD Ang II M U 5-aza M U Ctrl M U Hcy M U DM E (Fold of control) * * Methylation ratio(%) Suppl. Fig. III. Hcy stimulated EC senescence by restoring human telomerase reverse transcriptase (hTERT) activity via DNA hypomethylation. (A) qPCR analysis of relative telomerase length (T/S ratio in P2-P10 HUVECs treated with Hcy or not, 25 μM. (B) qRT-PCR analysis of mRNA expression of hTERT, hTR, TRF2 and hTP1 relative to PBS control with Hcy, 50 μM, for 72 hr in P4-P6 HUVECs. β-actin was an internal control. (C) Proportion of SA-β-gal–positive cells to PBS control and (D) qRT-PCR analysis of hTERT mRNA expression in HUVECs with Hcy (50 μM), angiotensin II (Ang II, 100 μM) or 5-aza (8 μM) with or without pre-supplementation with FA (100 μM), L-arginine (LAR, 50 μM) or SAM (100 μM). (E) MSP assay of hTERT methylation in HUVECs with Hcy, Ang II or 5-aza treatment. *P<0.05 vs. PBS control, # P<0.05 vs. Hcy, § P<0.05 vs. Ang II, ※ P<0.05 vs. 5-aza. Data are mean±SD (n=4 for T/S ratio assay; n=5 for qPCR, MSP and SA-β-gal staining assay, as well as 2 parallel samples were measured each time). A hTREThTRTRF2hTP mRNA relative level * B P2P4P6P8 P Ctrl T/S ratio * * * Hcy λ λ Ctrl FA LARLAR SAM Hcy FA+Hcy LAR+Hcy SAM+Hcy Ang II FA+Ang II LAR+Ang II SAM+Ang II 5-aza-CdR FA+5-aza-CdR LAR+5-aza-CdR SAM+5-aza-CdR  -gal positive rate (%) * * * * # # # & & ※ ※ hTERT mRNA expression ※ ※ * * ** ** Ctrl FA LARLAR SAM Hcy FA+Hcy LAR+Hcy SAM+Hcy Ang II FA+Ang II LAR+Ang II SAM+Ang II 5-aza-CdR FA+5-aza-CdR LAR+5-aza-CdR SAM+5-aza-CdR

Supplement Fig. IV Suppl. Fig. IV. Knockdown of SP1, CTCF and DNMT1 by si-RNA in HUVECs. qRT-PCR and Western blot analysis of expression of CTCF (A), SP1 (B) and DNMT1 (C), after P4 HUVECs were transfected with 40-nM siRNA for SP1, CTCF and DNMT1 or scramble siRNA control (si-Ctrl) for 12 hr. Data are mean±SD (n=4). TATA binding protein (TBP) was an internal control. *P<0.05 vs si- Ctrl transfection. SP1 Si-CtrlSi-SP SP1 mRNA relative level Si-CtrlSi-SP1 ** A B C DNMT1 Si-CtrlSi-DNMT DNMT1 mRNA relative level Si-CtrlSi-DNMT1 ** Si-CtrlSi-CTCF CTCF mRNA relative level CTCF Si-CtrlSi-CTCF * TBP

Hcy Ctrl CTCF IP: CTCF IgG SP1 Input SP1 CTCF IgG CTCF IgG Hcy Ctrl Hcy Ctrl IP: SP1 IgG Supplement Fig. V A B C Supplement Fig. V Interaction analysis between SP1 and CTCF by co-immunoprecipitation in P4 HUVECs treated with or without Homocysteine (50 μmol/L) for 72 hr. The experiments were performed in 4 independent times.

A NOX4 mRNA expression CtrlHHcyCtrlHHcy 4 W 8 W Supplement Fig. VI NOX4 DIPA NOX4 DIPA 4 W 8 W Ctrl HHcy * * B Suppl. Fig. VI. NOX4 expression in aortic intima of hyper-Hcy (HHcy) mice. C57 mice were fed standard chow with or without 2% methionine for 4 and 8 weeks (n=8, in each group). (A) qRT- PCR analysis of NOX4 mRNA level in aortic intima. Normalization was to mouse β-actin level. *P<0.05 vs Ctrl group. (B) Representative immunohistochemical staining of NOX4 (red) and nuclei (blue) in cross sections of aortas from HHcy and control mice.