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Fig. S1 Senescent endothelial cells exhibit enhanced Arg-II expression and activity. (A) SA-β-gal staining in young (Y) and senescent (S) HUVECs. (B) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1 levels, and endothelial inflammation markers VCAM1 and ICAM1 expression. Tubulin served as protein loading control. (C) Immunoblotting analysis of Arg-II protein levels and arginase activity. ***p < 0.005 vs. Y group. Scale bar = 0.2 mm.

Fig. S2 Overexpression of Arg-II gene in young cells induces eNOS uncoupling, endothelial senescence, and inflammation. Young endothelial cells were transduced with empty rAd/CMV vector as control (con) or rAd/CMV-Arg-II to overexpress Arg-II. (A) Immunoblotting analysis of Arg-II overexpression. (B) DHE staining for detection of O2.- and DAF-2DA staining for detection of NO, and effect of the eNOS inhibitor L-NAME (1 mmol/L, 1 h). Bar graphs show quantifications of DHE and DAF-2DA signals. (C) SA-β-gal staining. Bar graphs show quantifications of percentage of SA-β-gal positive cells. (D) Immunoblotting analysis of senescence markers p53-S15, p53, and p21Cip1 levels, and endothelial inflammation markers VCAM1 and ICAM1 expression. Tubulin serves as loading control. Bar graphs show quantifications of the markers. **p < 0.01, ***p < 0.005 vs. control; p < 0.05 vs. Arg-II. Scale bar = 0.2 mm.

Fig. S3 Enzymatic activity dependence of the Arg-II-promoted endothelial aging in young cells. The young endothelial cells were transduced with empty rAd/CMV vector as control (con), rAd/CMV-Arg-II to overexpress wild-type Arg-II or with rAd/CMV-Arg-II (H160F) to overexpress the inactive Arg-II mutant. (A) Immunoblotting analysis of Arg-II overexpression. (B) DHE staining for detection of O2.- and DAF-2DA staining for detection of NO. Bar graphs show quantifications of DHE and DAF-2DA signals. (C) SA-β-gal staining. Bar graphs show quantifications of percentage of SA-β-gal positive cells. (D) Immunoblotting analysis of p53-S15, p53, p21Cip1 levels, VCAM1, and ICAM1 expression. Tubulin serves as loading control. Bar graphs show quantifications of the markers. *p < 0.05, **p < 0.01, ***p < 0.005 vs. control; ††<0.01, †††p < 0.005 vs. Arg-II. Scale bar = 0.2 mm.

Fig. S4 Silencing Arg-II prevents S6K1-induced Arg-II expression and arginase activity. Young HUVECs were first transduced either with rAd/U6-LacZshRNA as control or rAd/U6-Arg-IIshRNA. Four days post transduction with rAd/U6-shRNA, cells were then transduced either with rAd/CMV as control (con) or rAd/CMV-HA-S6K1ca (a constitutively active S6K1 mutant). Experiments were performed on day two post 2nd transduction. Cells were serum starved with 0.2% FCS-RPMI 12 h prior to experiments. Blots above reveal the immunoblotting analysis of HA-tagged S6K1ca and Arg-II with anti-HA and anti-Arg-II antibody, respectively. Bar graphs below show quantifications of Arg-II/tubulin protein level, arginase activity, and Arg-II/GAPDH mRNA levels as analysed by qRT-PCR. ***p < 0.005 vs. control; †††p < 0.005 vs. S6K1ca.

Fig. S5 Deficiency in Arg-II gene in mice (Arg-II-/-) improves eNOS function in aging. (A) Immunofluorescence confocal microscopy showing en face detection of aortic endothelial O2.- (DHE staining) and NO production (DAF-2DA staining) in young (2–3 months) and old (23–24 months) WT and Arg-II-/- mice followed by counterstaining with DAPI for endothelial nuclei. (B) Bar graphs show quantifications of DHE and DAF-2DA signals. **p < 0.01, ***p < 0.005 vs. young WT mice; p < 0.05, †††p < 0.005 vs. old WT mice. Scale bar = 100 μm.

Fig. S6 S6K1 activity in aging is positively regulated by Arg-II. Immunoblotting analysis of S6-S235/S236 (p-S6) and total S6 in (A) aortas of young (2–3 months) and old (23–24 months) WT and Arg-II-/- mice and (B) senescent endothelial cells transduced with LacZshRNA as control or Arg-IIshRNA. Bar graphs below show quantifications of the above experiments. **p < 0.01, ***p < 0.005 vs. young WT mice or LacZshRNA group; †††p < 0.005 vs. old WT mice. (C) Scheme illustrating mutual positive crosstalk between S6K1 and Arg-II in endothelial aging.

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