Fig 2: Expression and activity production of NOS in WT and Tgß3 rats at 45 weeks of age. Effects of the ß3-AR overexpression in Tgß3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, L-VNIO, and 1400W) (e) and O2•- production with or without L-NMMA (f) from WT and Tgß3 rats. Data are expressed as mean ± SEM. n = 5-10. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: Effects of the inhibitor of SRPK1 on eNOS activity in HUVECs. HUVECs were treated with different concentrations of SPHINX31 for 24 h. The levels of eNOS, phosphor-eNOS (Ser1177), Akt, phosphor-Akt (Ser473), and SRPK1 were determined by western blot (A) with the quantifications of the related proteins (B). After treatment with different concentrations (1, 2.5, and 5 µM) of SPHINX31 for 24 h, the immunofluorescence images of the NO probe were observed using 40 × fluorescence microscopy (C) with the quantifications of mean fluorescence intensity (D). The data are presented as the mean ± SD of three independent experiments. Significance was determined by one-way ANOVA with a least significant difference post hoc test. ns, p > 0.05; *, p < 0.05 vs Ctrl.

Fig 4: Activation of SRPK1-Akt-eNOS pathway in HUVECs treated with ICA ?. HUVECs were treated with different concentrations of ICA ? (1 and 2 µM) in the presence or absence of SPHINX31 (5 µM) for 24 h. The levels of eNOS, phosphor-eNOS (Ser1177), Akt, phosphor-Akt (Ser473), and SRPK1 were determined by western blot (A) with the quantifications of the related proteins (B). After the same treatment, the immunofluorescence images of the NO probe were observed using 40× fluorescence microscopy (C) with the quantifications of mean fluorescence intensity (D). The data are presented as the mean ± SD of three independent experiments. Significance was determined by one-way ANOVA with a least significant difference post hoc test. ns, p > 0.05; *, p < 0.05 vs Ctrl.

Fig 5: WS® 1442 normalizes eNOS acetylation level in PVAT. Male C57BL/6J mice were put on a NFD or HFD for 22 weeks starting at the age of 8 weeks. A subgroup of HFD animals were treated with WS® 1442, p.o., during the last 4 weeks of HFD feeding. Immunoprecipitation (IP) was performed with an anti-eNOS antibody using PVAT samples followed by immunoblotting (IB) with antibodies against eNOS or acetyl-lysine respectively. The band detected with the antibody against acetyl-lysine at the level of eNOS was considered acetyl-eNOS. In (A) the blots shown are representative of six independent experiments. (B) Results of densitometric analyses for acetyl-eNOS normalized to total eNOS; n = 6. (C,D and E) Expression of SIRT1 and NAMPT at the protein (C and D) or mRNA (E) levels was analysed by western blotting and qPCR respectively. In (C) the blots shown are representative of six independent experiments. (D and E) Results of densitometric analyses are shown; n = 6. Columns represent mean ± SEM. *P < 0.05.