a! b! c! Diamet (μm) 2 2 1 WT Nogo-A/B-deficient -9-8 -7 - -5-4 PE (LogM) Diamet (μm) 2 2 1-12-11-1 -9-8 -7 - -5 U-419 (LogM) Diamet (μm) 2 2 1-8 -7 - -5 S1P (LogM) d! WT! Nogo-A/B-deficient! MLE! enos! β-actin! enos/β-actin (a.u.) 1.5 1..5. WT Nogo-A/B-deficient e! WT! Nogo-A/B-deficient! nnos! β-actin! Aorta! nnos/β-actin (a.u.) 1.5 1..5. Supplementary Fig. 1! Nature Medicine: doi.138/nm.3934
b! 15 1 5 3 1 4 e r 4 5 3 2 1 4 ** 8 18 15 ** 2 2 22 pmol*mg-1*ml-1 25 pmol*mg-1*ml-1 Nogo-A/B-deficient pmol*mg-1*ml-1 Total camide (pmol*mg-1*ml -1) WT 2 dh d! c! ontrol! myriocin.3 nm! myriocin 1 nm! 1 5 8 4 2 dh -S 1P dh Sp h Sp h S1 P e! Sphinganine inhibition (%) a! 3 9.1.1 1 1 1 1 Dose (nmol/l) myriocin 3 nm!.3 1 3 1 3 1 3! HUVE + myriocin (nm)!! SM! PS.1 e nin ga hin de! Sp ami r e myriocin 1 nm! f! microsomes! WT! soluble! fraction! Ng-A/BNg-A/Bdeficient! WT! deficient!! myriocin 3 nm! L! alnexin! HSP9! Supplementary Fig. 2! Nature Medicine: doi.138/nm.3934
Nature Medicine: doi.138/nm.3934
a! Nogo-A/Bf/f! b! VE-cadhin! αsma! HSP9! c! VE-cadhin! IB4! αsma! HSP9!.8.4. Supplementary Fig. 4! Nature Medicine: doi.138/nm.3934 2. 1..8.4. g! f! E VSM 2. 1..8.4. E VSM Nogo-A/Bf/f SM-Nogo-A/B-deficient 1..8.4. h! 1 % Initial diamet e! Nogo Nogo-A/Bf/f E-Nogo-A/B-deficient 1. Nogo d! VE-cadhin α-sma! αsma E-! Nogo-A/B-deficient! α-sma! SM-! Nogo-A/B-deficient! 5 μm! Nogo-A/Bf/f E-NogoA/B-deficient SM-NogoA/B-deficient 2-9 -8-7 - -5-4 Phe (LogM)
SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. The loss of Nogo-B does not affect the vasoconstriction in response to pharmacological agents. oncentration-response curves of WT and Nogo-A/B-deficient MA in response to (a) PE, (n = 8 WT vs. n = 7 Nogo-A/B-deficient), (b) U-419, (n = 5 p group) and (c) S1P (n = 1 WT vs. n = Nogo-A/B-deficient). WB analysis and relative quantification of (d) enos on MLE from WT and Nogo-A/Bdeficient mice, n = 4 isolations p group, and (e) nnos on WT and Nogo-A/B-deficient aortas (n = 4 aortas from 4 mice p group). β-actin was used as loading control. Data we expressed as the mean ± s.e.m. **P<.1 compared to WT group. Statistical significance was detmined by two-way ANOVA followed by Bonfroni s post-test (a c) and unpaired t-test (d e). Supplementary Figure 2. Sphingolipids levels in vascular SM and SPT assay in E. WT and SM-Nogo- A/B-deficient (a) total camide levels and (b) individual sphingolipid species measured by L/MS as described in Methods. n = 4 independent VSM isolation/group; five mice we used for each VSM isolation. Mean ± s.e.m. (c) Pharmacological inhibition of SPT activity by increasing concentrations of myriocin in HUVE. The inhibition was assessed using [ 3 H]sine as substrate in endothelial cell lysates as described in Methods. amide-8 (camide), sphinganine, phosphatidylsine (PS) and sphingomyelin-8 (SM). (d) Extracted lipids we separated by TL and [ 3 H]sphinganine was detected by using TL scann (TL analyz RITA, Raytest Straubenhardt) and reading values we quantified using a standard curve of [ 3 H]sine. (e) Inhibition of SPT activity, expressed as pcent of the control, by increasing concentrations of myriocin. (f) WB analysis for Nogo-B, SPTL, alnexin and HSP9 in microsomes isolated from WT and Nogo-A/Bdeficient mouse lung. Unpaired t-test was pformed to evaluate the statistical significance. Supplementary Figure 3. Effects of W14 and myriocin on vascular tone regulation. (a) Ach cumulative concentration-response curve in WT mesentic arties treated with myriocin (.3 mg/kg i.p.) or vehicle in presence of L-NAME, (left, n = 7 p group), indomethacin, (middle, n = 13 vehicle vs. n = 9 myriocin), and their combination, (right, n = 5 vehicle vs. n = myriocin). (b) oncentration-response curve of S1P in MA from WT and Nogo-A/B-deficient mice incubated with L-NAME or vehicle. n = 4 p group. (c) WT mesentic arties treated with W14 (1 nm, 45 min) or vehicle, we incubated with L-NAME and the increase in the basal tone was measured as intnal diamet decrease ov the baseline. (n = 11 ctrl vs. n = 5 W14 1 nm). (d) The active diamet of WT mesentic arties treated with diffent concentrations of W14 or vehicle in response to stepwise increase in intraluminal pressure. (n = 12 ctrl vs. n = 5 W14 1 nm and 1 μm). (e) Vasoconstriction induced by PE following W14 treatment of WT mesentic arties. (n = 8 ctrl vs. n = 9 W14 1 nm). (f) Ach-induced vasodilation in WT mesentic arties following W14 (1 nm) treatment. Given that basal and PE-stimulated tone is diffent aft W14 treatment, the following data are expressed as Nature Medicine: doi.138/nm.3934
absolute intnal diamet (μm) of MA. (n = 21 ctrl vs. n = 7 W14 1 nm). (g) In anoth set of expiments, aft incubation with W14, Ach cumulative concentration-response curve was pformed in presence of: L- NAME (left, (n = 8 ctrl vs. n = 5 W14 1 nm),.indomethacin (middle, n = 7 p group) and their combination (right, n = p group). (h) Schematic summary of myriocin and W14 effects on the vascular tone. Data are expressed as the mean±s.e.m. *P<.5; **P<.1 compared to vehicle. Statistical significance was detmined by two-way ANOVA followed by Bonfroni s post-test (a b, d g) and unpaired t-test (c). Supplementary Figure 4: haractization of the re-mediated excision of Nogo-B in E and VSM of E- Nogo-A/B-deficient and SM-Nogo-A/B-deficient mice. (a) IF staining of MA cross-sections for: (top panels) Nogo-B (red) and α-smooth muscle actin (αsma; green) in NogoA/B f/f MA; (central panels) Nogo-B (red) and E with isolectin-b4 (IB4, green) in SM-Nogo-A/B-deficient MA; and (bottom panels) Nogo-B (red) and αsma (green) in SM-Nogo-A/B-deficient MA. (b) Westn blot analysis for Nogo-B in E isolated from NogoA/B f/f and E-Nogo-A/B-deficient lungs. (c) Westn blot analysis for Nogo-B in VSM isolated from NogoA/B f/f and SM-Nogo-A/B-deficient thoracic aortas as described in Methods. Heat shock protein 9 (HSP9) was used as loading control. Vascular endothelial cadhin (VE-cadhin) and αsma we used as lineage marks of E and smooth muscle cells/fibroblasts, respectively. (d) RT-PR for Nogo-B in E isolated from NogoA/B f/f and E-Nogo-A/B-deficient lungs; RT-PR for (e) αsma and (f) VE-cadhin in endothelial and smooth muscle cells mrna isolated from NogoA/B f/f and SM-Nogo-A/B-deficient thoracic aortas as described in Methods. (g) Nogo-B expression of thoracic aorta VSM mrna isolated from NogoA/B f/f and SM-Nogo-A/B-deficient mice (h) PE-concentration-response curves of Nogo-A/B f/f, E-Nogo- A/B-deficient and SM-Nogo-A/B-deficient MA. Nature Medicine: doi.138/nm.3934