Extended Data Fig. 6: Chondrocytes do not depend on FAO.

a, Quantification of glycolytic rate, oxygen consumption and palmitate oxidation in periosteal cells (PC, n = 5 biologically independent samples), skeletal stem cells (SSC, n = 3 biologically independent samples), growth plate-derived chondrocytes (GCH, n = 3 biologically independent samples for oxygen consumption, n = 4 biologically independent samples for glycolysis and palmitate oxidation), rib chondrocytes (RCH, n = 5 biologically independent samples for oxygen consumption, n = 4 biologically independent samples for glycolysis and palmitate oxidation), calvarial osteoblasts (COB, n = 5 biologically independent samples) and trabecular osteoblasts (TOB, n = 5 biologically independent samples). b, t-Distributed stochastic neighbour embedding (t-SNE) plot of 20,896 non-haematopoietic cells (mixed bone and bone marrow fractions, n = 6 mice) based on single-cell RNA-seq data, annotated post hoc and coloured by clustering (top) or by expression (ln(TP10K)) of selected genes (bottom). c, Expression (row-wide Z score of ln of average TP10K; single-cell RNA-seq) of FAO- and glycolysis-related genes (rows) in the cells of each cluster (columns). d, RT–qPCR analysis of genes involved in glycolysis (Glut1 (also known as Slc2a1), Pfkfb3 and Ldha; n = 6 independent samples for Glut1 and Pfkfb3 in cartilage, n = 9 independent samples for Glut1 and Pfkfb3 in bone, n = 8 independent samples for Ldha) and FAO (Cpt1a, Acadm and Acadl; n = 8 independent samples) in mouse growth plate cartilage and cortical bone biopsies (relative to Actb). e, Analysis of adjacent histological sections of a growth plate and fracture callus (PFD7) of mice injected intravenously with a fluorescent fatty acid (Red-C12) or glucose (2-NBDG) (representative images of n = 3 mice). Scale bars, 100 μm in growth plate images, 50 μm in fracture callus images. f, Immunofluorescence analysis of a fracture callus (PFD7) of a mouse injected intravenously with a fluorescent fatty acid (Red-C12) and stained for SOX9 (left; cartilage area shown) or COL1 (right; trabecular bone area shown) (representative images of n = 3 mice). Scale bars, 50 μm. g, Histological visualization and quantification at PFD7 of CAG-DsRed⁺ skeletal stem cells (SSC), transduced with shCpt1a or shScr and transplanted at the fracture site on PFD0 (n = 3 mice). Dotted lines delineate cortical bone ends. h, Quantification of number of live and dead cells in cultures of periosteal cells, growth plate-derived chondrocytes and calvarial osteoblasts after 48 h of exposure to etomoxir (n = 3 biologically independent samples). Data are mean ± s.e.m.; one-way (a) or two-way (h) ANOVA with Bonferroni post hoc test, two-tailed Student’s t-test (d, g).

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