Extended Data Fig. 6: Effects of modulating GLS activities on metabolic risk factors and architectural regulation of the ECM.

(a) Liver and (b) plasma Gln (left panel) and Glu (right panel) levels and (c) muscle GGR in Apoe^−/− mice infected with the AAV-Gls2^Ovex or AAV-empty controls and fed an atherogenic diet for 12 weeks (n = 8–9 mice per group; P values displayed; two-tailed t test). (d) From left to right: plasma cholesterol, TG, ALT, urea levels and blood monocyte counts in these mice (n = 8–9 mice per group; P values displayed; two-tailed t test). (e) Aortic GGR in a subgroup of these mice (n = 5 mice per group; P = 0.05; two-tailed t test). (f) Aortic GGR in Ldlr^−/−Gls2^−/− and Ldlr^−/− control mice (n = 7–9 mice per group; P < 0.0001; two-tailed t test). (g) Volcano plot of differentially expressed genes (up, red and down, blue) between Ldlr^−/−Gls2^−/− and Ldlr^−/− aortas. (h) Representative high-resolution Fourier transform infrared (FTIR) images based on the aliphatic chain hydrocarbon (ACH) contribution by FTIR spectroscopy (left panel) or hyperspectral images represented as three color (RGB) composite images (middle panel) recorded in the aortic sinus of atherogenic diet-fed Ldlr^−/−Gls2^−/− and Ldlr^−/− mice. Scale bar, 200 mm. Quantification of the lipid from aliphatic chain hydrocarbon band (represented in red) and the protein from Amide I band (represented in green) in the aortic sinus of these mice (right panel) (n = 3 mice with up to 3 measures per images; P = 0.002; two-tailed t test). (i) Surface plot reconstitution of spectral density plot from images created using factor analysis. (j) Representative infrared images after attenuated total reflectance (ATR) showing the absorbance band centered at 1648 cm^-1 (Amide I vibrations) and 1000-1200 cm^-1 (proteoglycan vibrations) in the aortic media of these animals. Scale bar, 50 mm. (k) Representative atomic force microscopy (AFM) 3D pictures of the aorta of these mice using a Peak Force QNM mode. (l) Verhoeff-Van Gieson (VVG) staining (scale bar, 200 mm) and quantification of elastic lamina area (expressed as arbitrary unit, a.u) in the proximal aorta of atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice (n = 5 mice per group; P = 0.02; two-tailed t test). Data from individual mice are shown and values are given as the mean ± s.e.m. of one independent experiment. Two-tailed Student’s t-tests were used.