Figure 4.

Protein restriction up-regulates cystathionine γ-lyase and H₂S levels in endothelial cells. (A and B) Representative images of inferior vena cava (IVC) immunofluorescence staining of CBS (A) or CGL (B). (C–H) Western blots (C, E, G) and quantitation (D, F, H) of CGL and CBS from whole thoracic aorta (C and D), aortic endothelial cells (ECs) (E and F), or IVC ECs (G and H) isolated from LDLr^−\− mice after 1 week on the indicated diet; Ponceau stained membranes were used as loading controls and CGL^−/− ECs were used as a control for CGL antibody specificity (E). (I–M) LDLr^−\− mice in the indicated treatment groups (HFHC, PR-HFHC, or PR-HFHC + PAG) were harvested after one week. (I and J) Whole thoracic aorta gene expression of CGL (I) and ATF4 (J); one-way ANOVA with Dunnet’s multiple comparisons test vs. HFHC control group. (K–M) Flow cytometric analysis of single cell isolates from lung after staining with CD31 and P3 (fluorescent H₂S probe). (K) Representative dot plots from the indicated groups with CD31/P3 double positive cells in the box. (L and M) Fold change, relative to HFHC group, in mean fluorescent intensity of P3 (L) and frequency (M) of CD31/P3 double positive cells; HFHC and PR-HFHC, n = 10/group; PR-HFHC+PAG, n = 4/group; Kurskal–Wallis test with Dunnett’s multiple comparisons test. All data expressed as mean ± SEM; *P < 0.05, **P < 0.01.