Figure 1. RANKL changes cellular metabolism of differentiating osteoclasts. (A) Experimental setup of RNA-seq workflow. BMCs were stimulated with M-CSF for 72 hours and then replated with M-CSF/RANKL for 48 hours in medium lacking either glutamine or glucose. (B) Venn diagram depicting the overlap between downregulated genes (FC<−1.5 and FDR<0.05) in cells stimulated as shown in (A) compared with cells cultivated in the control medium (n=3). (C) Top 10 enriched pathways with >5 genes/pathway from 1197 overlapping genes from (B) assessed by GO enrichment analysis. Colour denotes FDR and size of the shape denotes affected genes in relation to all genes from a particular pathway. (D) Absolute abundance of TCA cycle intermediates in cells stimulated with either M-CSF or M-CSF/RANKL for 3 hours, 9 hours and 24 hours (n=4). (E) Schematic depicting the fate of U-¹³C labelled atoms derived from U-¹³C glucose. Light red circles indicate labelled C-atoms. (F) M+2 fraction of the total pool for analysed TCA cycle intermediates in cells cultivated in medium supplemented with U-¹³C glucose and stimulated with either M-CSF or M-CSF/RANKL for 6 hours (n=4). (G) Schematic depicting the fate of U-¹³C labelled atoms derived from U-¹³C glutamine. Light red circles indicate labelled C-atoms. (H) M+4 fraction of the total pool for analysed TCA cycle intermediates in cells cultivated in medium supplemented with U-¹³C glutamine and stimulated with either M-CSF or M-CSF/RANKL for 6 hours (n=4). The data are represented as means±SDs. One-way ANOVA with Šidàk’s correction (D, F, H). BMCs, bone marrow-derived cells; RANKL, receptor activator of nuclear factor κB ligand; TCA, tricarboxylic acid.