Extended Data Fig. 2: Effects of defective hepatic glutaminolysis on Gln-to-Glu cycle in atherosclerosis.

(a) Experimental outline. Apoe^−/− mice were infected with the AAV-Gls2^shRNA (Apoe^−/−Gls2^shRNA mice) or AAV-scrambled shRNA controls and fed an atherogenic diet for 12 weeks (left panel). mRNA expression of Gls2 in indicated tissues (values were normalized to m36B4 and expressed as arbitrary unit) (n = 8 mice per group; P values displayed; two-tailed t test) and Western blot of GLS2 protein in the liver of these mice (middle panel). GLS2 was also visualized by immunofluorescent staining in the hepatic periportal vein (right panel). Scale bar, 200 mm. (b,c) Tissue and (d) plasma Gln and Glu levels and GGR in these mice (n = 9-10 mice per group except liver tissue due to outlier analysis (Grubb’s test); P values displayed; two-tailed t test). (e) Overall survival curves of atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice after restraint stress exposure and expressed as the percentage of mice surviving at the times indicated. (f) Experimental outline. Ldlr^−/−Gls2^−/− and Ldlr^−/− mice were fed an atherogenic diet for 12 weeks (left panel). Western blot of GLS2 protein in the liver of these mice (right panel). (g) Tissue (n = 8-13 mice per group due to failure of some measurements; P values displayed; two-tailed t test) and (h) plasma (n = 13-20 mice per group from two separate experiments; P values displayed; two-tailed t test) Gln and Glu levels and GGR. Data from individual mice are shown and values are presented as the mean ± s.e.m. of at least one independent experiment unless otherwise stated on the corresponding panel legend. Two-tailed Student’s t-tests were used.