Extended Data Fig. 3: Lesional monocyte infiltration in Gls2-deficient mice independent of systemic inflammation.

(a) Leukocyte population gating strategy and (b) percentage measured by flow cytometry from the blood of atherogenic diet-fed Apoe^−/−Gls2^shRNA compared to controls (n = 9-10 mice per group; P values displayed; two-tailed t test) or Ldlr^−/−Gls2^−/− mice compared to respective controls ( = 13-18 mice per group; P values displayed; two-tailed t test). (c) Tracking recruitment of Ly6C^hi monocytes to atherosclerotic plaques two days after monocyte labeling with latex beads in atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice. Representative flow cytometry plots (left panel) and quantification (right panel) of latex+ monocytes that have infiltrated the aortic arch of atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice. (d) Representative pictures (left panel) and quantification of latex beads (indicated by arrows) localized within atherosclerotic lesions and expressed as the number of beads per cross section (right panel) (n = 6 mice per group; P values displayed; two-tailed t test). Scale bar, 200 mm. (e) Venn diagram highlighting the common and specific Gls2-dependent regulated genes between the Ldlr^−/− and Apoe^−/− atherogenic strains. (f) Differentially expressed genes between Gls2-deficient mice and controls on atherogenic Apoe^−/− background were subjected to gene set enrichment analysis (GSEA). The top 4 up- and downregulated pathways are displayed (n = 3-4 mice per group; log₂ fold change was used to assess statistical significance with two-tailed t test, with P < 0.05). (g) RNAseq analysis focusing on the previously identified hepatic energy-sensing signature governing monocyte homeostasis in Gls2-deficient mice and controls on atherogenic Ldlr^−/− and Apoe^−/− background. (h) Quantification of myeloid cells in the bone marrow and spleen and Kupffer cells (KCs) at the end of the study period in atherogenic diet-fed Apoe^−/−Gls2^shRNA and control mice (n = 4–5 mice per group; P values displayed; two-tailed t test). (i) Expression of chemokines and interleukins in the liver (left panel), plasma (middle panel) and BM fluid (right panel), assessed by multiplex assays. Data from individual mice are shown and values are given as the mean ± s.e.m. of at least one independent experiment. Two-tailed Student’s t-tests were used.