Fig. 3. CMA increases linoleic acid metabolism upon HSC activation.

a, Top lipid metabolism pathways identified from enrichment analysis of metabolites as different between control and L2AKO LSK cells 8 days post 5FU injection. b, Heat map of substrate and metabolite abundance in the linoleic acid and α-linolenic metabolism in the indicated conditions. c, Scheme of linoleic and α-linolenic fatty acid metabolism pathway showing the increased (red arrow) and decreased (green arrow) metabolites in L2AKO LSK cells comparing with Ctrl cells in 5FU-activated conditions. d, Levels of substrates (black, gray) and downstream metabolites (green) of the linoleic acid and α-linolenic metabolism relative to those in unstimulated control cells. n= 9 mice in 3 different experiments. Significant differences with control untreated are marked with * and between L2AKO basal and 5FU treated with †. e, Total colony numbers (left) and number of immature, multipotent colony-forming unit-granulocyte/ erythroid/macrophage/megakaryocyte CFU-GEMM (right) in the first plating from Ctrl and L2AKO HSC treated or not with the FADS2 inhibitor SC-26196 (SC). n=3 independent experiments. f, Total colony numbers in the 3^rd plating (left) or number of CFU-GEMM colonies in the first plating (right) from Ctrl and L2AKO HSC treated or not with γ-linolenic acid (GLA). n=4 independent experiments. g, Predicted CMA-targeting motif in FADS2 (green) generated by acetylation of ⁴²K (g top) and analysis of the acetylation level of this peptide detected by mass spectrometry Ctrl and L2AKO LSK cells in basal and 5FU activated conditions (g bottom). n=3 mice. h,i, Immunoblot for Flag of Ctrl (h) and L2AKO (i) ex vivo expanded HSC expressing Flag-myc tagged FADS2 wild type (VIDRK) or mutated as indicated at the top (VIDRQ or VIDRA) treated or not with NH₄Cl/Leupeptin (N/L). For gel source data, see supplementary Figure 1. j, Oxygen consumption rate (OCR) in Ctrl and L2AKO LSK cells at day 8 after 5FU injection. Responses to addition of oligomycin (Oligo), FCCP and rotenone (Roteno) are shown. n=5 independent experiments. k, Quantification of mitochondrial respiration from fatty acid β-oxidation (etomoxir-sensitive) in Ctrl and L2AKO LSK cells. n=5 independent experiments. l, Total colony numbers in 3^rd plating from both Ctrl and L2AKO HSC treated or not with Methyl-pyruvate (5mM) starting from the 1^st plating. n=7 (None) and 4 (Methyl-pyruvate) independent mice. m, Quantification of MFI of ROS⁺ cells (left) and total colony numbers (right) in the 3^rd plating of Ctrl or L2AKO HSC untreated or supplemented with NAC from the 1^st plating. n=7 (None) and 4 (NAC) mice. n, Ratio of γ-linolenic acid (GLA) and linoleic acid (LA) in LSK cells from young and old mice calculated from the metabolomics data. n= 9 mice in 3 different experiments. o, Ratio of K42 acetylated and total FADS2 peptide in LSK from young and old mice calculated from the mass spectrometry analysis. n= 9 mice in 3 different experiments. p, Total colony numbers (left) and number of CFU-GEMM colonies (right) in the colony formation assay with HSC cells from old (22m) mice daily injected with either saline or GLA (1mg/kg bw) for 7 weeks. Representative images of wells with Ctrl or GLA treated cells are shown on the top. n=5 mice per group. q, LTC-IC assay according to GEMM colonies from Ctrl and GLA treated human CD34⁺ cells from old (>65 year) multi-myeloma patients. n=2 patients. Two-way ANOVA test followed by Tukey’s (d,f) or Sidak (e,l,m) multiple comparison post-hoc test, unpaired t-tests (g,k,n,o,p), time point paired t-test (j), and Chi-square test (q) were used for the statistics. P<0.05 (*), 0.01 (**), 0.001(***), 0.0001(****). ns: no statistical significance.