Extended Data Fig. 8: Lipids regulate SOX9 through FOXO signalling.

a, Heat map showing differential expression of cartilage-related genes in C3H10T1/2 cells exposed for different times to SD versus control medium, as determined by RNA-seq (n = 3 replicates). b, Volcano plot showing significantly enriched and depleted mRNAs in C3H10T1/2 cells exposed for 3 or 6 h to SD versus control medium, as determined by RNA-seq (n = 3 replicates). c, Top 10 most significantly enriched transcription factor motifs with normalized enrichment scores (NES) in C3H10T1/2 cells exposed for 3 h (left) or 6 h (right) to SD versus control medium, as determined by i-cisTarget analysis on the 100 most-significantly increased mRNAs (n = 3 replicates). Motif shown on top is the Hmga1 motif for 3 h and the Atf4 motif for 6 h. d, Confocal microscopy of C3H10T1/2 cells stained for FOXO1 after exposure of cells for 3 h to SD or LRS medium in the presence of vehicle (EtOH), oleate (60 μM) or PUFA (representative images of two independent experiments). Scale bars, 20 μm. e, Nuclear FOXO activity in C3H10T1/2 cells exposed for 3 h to control, SD or LRS medium (n = 5 independent experiments). f, Nuclear FOXO activity in skeletal stem cells exposed for 3 h to control medium, LRS medium or LRS medium supplemented with PUFA (n = 3 biologically independent samples). EtOH was used as vehicle control. g, Occupancy of FOXO1 at the Sox9 promoter of Cas9-expressing C3H10T1/2 cells transduced with sgFoxo1, sgFoxo3a or sgScr, exposed for 3 h to control or SD medium, as determined by ChIP–qPCR (n = 3 independent experiments). h, Flow cytometric quantification of total SOX9 levels in C3H10T1/2 cells (n = 4 independent experiments for control and serum deprivation, n = 3 independent experiments for LRS) and skeletal stem cells (n = 3 biologically independent samples) exposed for 24 h to control, SD or LRS medium supplemented with 1 μM AS1842856 or vehicle (DMSO). i, Immunoblot detection of total SOX9 in Cas9-expressing C3H10T1/2 cells transduced with inducible sgFoxo1 and sgFoxo3a (sgFoxo1/3a) or with sgScr, exposed for 6 h to control, SD or LRS medium in the presence or absence of doxycycline (dox; 250 ng ml⁻¹), with β-actin as loading control (n = 2 independent experiments). j, Flow cytometric quantification of total SOX9 levels in skeletal stem cells transduced with shFoxo1 and shFoxo3a (shFoxo1/3a) or with shScr, exposed for 24 h to control, SD or LRS medium (n = 5 biologically independent samples). k, Histological visualization and quantification of FOXO3a-expressing cells in the fracture callus at PFD7 of mice treated daily with GW9508 (10 nmol) or vehicle (0.2% DMSO in saline) at the fracture site (n = 5 mice). Scale bars, 500 μm. Dotted lines delineate cortical bone ends. l, Histological visualization and quantification in the fracture callus at PFD7 of CAG–DsRed⁺ skeletal stem cells (SSC), transduced with shFoxo1/3a or shScr and transplanted at the fracture site on PFD0 (n = 5 mice). Dotted lines delineate cortical bone ends. Data are mean ± s.e.m.; one-way ANOVA (e, f), two-way ANOVA (g, h, j) with Bonferroni post hoc test, two-tailed Student’s t-test (k, l). For gel source data, see Supplementary Fig. 1.

Source data