Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a ) Immunofluorescence analysis of bone graft periosteal cell tracing showing contribution to cartilage and bone (arrows: GFP + osteoblasts, arrowheads: GFP + osteocytes) in the graft callus at PFD14, while CD31 + blood vessels (red) are mainly host-derived (representative images of 4 mice). Scale bars, 50μm. ( b ) Immunofluorescence analysis of a bone autograft section revealing the interconnected periosteal callus and skeletal muscle vasculature at PFD7 (representative image of 3 mice). Scale bar, 200μm. ( c ) Schematic representation of the autograft model with filter. ( d ) Immunohistochemical analysis and quantification of callus vascularization at PFD7 when a filter with 30μm (filter 30; arrows indicate blood vessels passing through filter pores) or 0.2μm (filter 0.2) pore size was placed in between muscle and graft (n=4 mice for control and filter 30, n=5 mice for filter 0.2). Scale bars, 50μm in detail images, 200μm in other images. ( e ) Visualization and quantification of early chondrogenic cells in the callus of grafts with or without a filter (0.2μm) at PFD7 by immunofluorescence for SOX9 (n=7 mice). Scale bars, 50μm. ( f ) Visualization and quantification of cartilage in the callus of autografts with and without filter (0.2μm) at PFD14 by immunofluorescence for collagen type 2 (COL2) (n=4 mice for control, n=6 mice for filter 0.2). Scale bars, 500μm. b: bone, c: cartilage, f: filter, g: graft, h: host, m: muscle, pc: periosteal callus. Mean ± s.e.m. One-way ANOVA with Bonferroni post-hoc test ( d ), two-tailed Student’s t-test ( e , f ).

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Immunofluorescence, Derivative Assay, Immunohistochemical staining, Pore Size, Control, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a , b ) Immunoblot detection of total SOX9 in C3H10T1/2 cells exposed for 24 hours to control or CND medium ( a ) or to different nutritional stresses ( b ), with β-actin as loading control (n=2 independent experiments). ( c ) Chondrogenic differentiation of periosteal cells in control or SD medium, assessed by visualization of chondrogenic matrix deposition (Alcian Blue staining) and quantification of Sox9 , Col2a1 and Acan mRNA levels (relative to Actin , n=6 biologically independent samples). ( d , e ) Immunoblot detection of total SOX9 in C3H10T1/2 cells exposed for 6 hours to control, SD or SD medium supplemented with increasing concentrations of oleate ( d ) or to LRS medium ( e ), with β-actin as loading control (n=2 independent experiments). ( f ) Chondrogenic differentiation of periosteal cells in control, LRS, SD or SD medium supplemented with 60μM oleate (OL), assessed by Alcian Blue staining and quantification of Col2a1 and Acan mRNA levels (relative to Actin , n=6 biologically independent samples). ( g ) Flow cytometric quantification of total SOX9 levels in periosteal cells exposed for 24 hours to control, SD or LRS medium supplemented with 100μM GW9508 (FFAR1/4 agonist) or vehicle (DMSO) (n=3 biologically independent samples). ( h ) Histological visualization (Safranin O staining) and quantification of cartilage and woven bone in the callus at PFD7 of mice treated daily with GW9508 (10nmol) or vehicle (0.2% DMSO in saline) at the fracture site (n=5 mice). Scale bars, 500μm. Mean ± s.e.m. Two-tailed Student’s t-test ( c , h ), one-way ANOVA ( f ) or two-way ANOVA ( g ) with Bonferroni post-hoc test. For gel source data, see .

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Western Blot, Control, Staining, Saline, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a ) Immunoblot detection of nuclear SOX9 in C3H10T1/2 cells and periosteal cells exposed for 24 hours to control or CND medium, with Lamin A/C as loading control (n=2 independent experiments). ( b ) mRNA levels of Sox9 and Col2a1 in periosteal cells exposed for the indicated times to control or CND medium (relative to control, n=3 biologically independent samples). ( c ) mRNA levels of runt-related transcription factor 2 ( Runx2 ; osteogenic lineage), peroxisome proliferator-activated receptor γ ( Pparg ; adipogenic lineage) and MyoD (myogenic lineage) in periosteal cells exposed for 48 hours to control or CND medium (relative to control, n=3 biologically independent samples). ( d ) mRNA levels of Sox9 in C3H10T1/2 cells exposed for the indicated times to control or SD medium (relative to control, n=3 independent experiments). ( e ) Immunoblot detection of total SOX9 in C3H10T1/2 cells exposed for different durations to control or SD medium, with β-actin as loading control (n=2 independent experiments). ( f ) Immunoblot detection of nuclear and cytoplasmic SOX9 in C3H10T1/2 cells exposed for 6 hours to control or SD medium, with Lamin A/C or β-actin as loading control (n=2 independent experiments). ( g ) Immunoblot detection of SOX9 in total cell protein extracts of C3H10T1/2 cells exposed for 6 hours to control medium, SD medium or SD medium supplemented with different concentrations of the transcription inhibitor Actinomycin D (Act. D) or the translation inhibitor cycloheximide (CHX). Detection of β-actin was used as loading control (n=2 independent experiments). ( h ) mRNA levels of Runx2 , Pparg and MyoD in C3H10T1/2 cells exposed for the indicated times to control or SD medium (relative to control, n=3 independent experiments). ( i ) Immunoblot detection of nuclear SOX9 in periosteal cells exposed for 24 hours to control or SD medium with Lamin A/C as loading control (n=3 biologically independent samples). ( j ) Osteogenic differentiation of periosteal cells in control or SD medium, assessed by visualization of mineral deposits (Alizarin Red staining) and quantification of Ocn mRNA levels (relative to Actin , n=3 biologically independent samples). ( k ) Immunoblot detection of SOX9 in total cell protein extracts of C3H10T1/2 cells (in control or SD medium), periosteal cells and growth plate-derived chondrocytes transduced with shSOX9 or shSCR, with β-actin as loading control. A longer exposure time was used for SOX9 detection in C3H10T1/2 cells and periosteal cells compared to chondrocytes in order to visualize any remaining protein in the shSOX9 conditions (n=2 independent experiments for C3H10T1/2 cells, n=3 biologically independent samples for periosteal cells, growth plate-derived chondrocytes). ( l ) Quantification of cell viability of C3H10T1/2 cells, periosteal cells and growth plate-derived chondrocytes transduced with shSOX9 or shSCR, after 72 hours of exposure to control, SD or CND medium (n=3 independent experiments for C3H10T1/2 cells, n=3 biologically independent samples for periosteal cells, growth plate-derived chondrocytes). Mean ± s.e.m. Two-way ANOVA with Bonferroni post-hoc test ( b , d , h , l ), two-tailed Student’s t-test ( c , j ). For gel source data, see .

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Western Blot, Control, Staining, Derivative Assay, Transduction, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a - c ) Immunoblot detection of total SOX9 in C3H10T1/2 cells exposed for 6 hours to control medium, SD medium or SD medium supplemented with increasing concentrations of palmitate ( a ), with VLDL ( b ), or with PUFA ( c ). Detection of β-actin was used as loading control. EtOH was used as a vehicle control in a and c (n=2 independent experiments). ( d ) Histological visualization (by immunofluorescence for COL2) of chondrogenic differentiation of periosteal cells in pellet cultures in control, SD medium or LRS medium supplemented with vehicle (EtOH), oleate or PUFA (representative images of n=2 independent experiments). Scale bars, 100μm. ( e ) Osteogenic differentiation of periosteal cells in control, SD medium or LRS medium, assessed by visualization of mineral deposits (Alizarin Red staining) and quantification of Ocn mRNA levels (relative to Actin , n=3 biologically independent samples). ( f ) Flow cytometric detection and quantification of the percentage of SOX9 high cells and total SOX9 levels in C3H10T1/2 cells, periosteal cells and skeletal stem cells exposed for 24 hours to control, SD or LRS medium (n=4 independent experiments for C3H10T1/2 cells, n=4 biologically independent samples for periosteal cells, skeletal stem cells). Gating for SOX9 high cells was set to have approximately 10% SOX9 high cells in control conditions in each cell type. ( g,h ) Flow cytometric quantification of cell cycle ( g ) and apoptosis ( h ) in SOX9 low and SOX9 high subpopulations of C3H10T1/2 cells, periosteal cells and skeletal stem cells exposed for 24 hours to control, SD or LRS medium (n=3 independent experiments for C3H10T1/2 cells, n=3 biologically independent samples for periosteal cells, skeletal stem cells). ( i ) Histological visualization and quantification of early chondrogenic (SOX9 + ) and osteogenic (Col1a1-DsRed + ) cells in metatarsals cultured for 1 week in control medium, SD medium, or SD medium supplemented with PUFA or vehicle (EtOH) (n=6 biologically independent samples for control, SD and SD+veh, n=7 biologically independent samples for SD+PUFA). Scale bars, 50μm. ( j ) Histological visualization of mineralization by Von Kossa staining in metatarsals cultured for 1 week in control medium, SD medium, or SD medium supplemented with vehicle or PUFA (representative images of n=6 biologically independent samples for control, SD and SD+veh, n=7 biologically independent samples for SD+PUFA). Scale bars, 100μm. ( k ) Histological visualization (Safranin O staining) and quantification of cartilage and woven bone in the callus at post-fracture day 7 of mice treated daily with free fatty acids (FFA; 20μl corn oil) or sham injection (saline) at the fracture site (n=5 mice). Scale bars, 500μm. ( l ) Flow cytometric quantification of total SOX9 levels in C3H10T1/2 cells or skeletal stem cells exposed for 24 hours to control, SD or LRS medium supplemented with 100μM GW9508 or vehicle (DMSO) (n=3 independent experiments for C3H10T1/2 cells, n=3 biologically independent samples for skeletal stem cells). ( m ) Visualization and quantification of diffusion of a fluorescent fatty acid (FL-C16) and fluorescent glucose (2-NBDG) in collagen gels seeded with periosteal cells (5 million/ml) (n=3 biologically independent samples for FL-C16, n=5 biologically independent samples for 2-NBDG). Scale bars, 500μm. ( n , o ) Visualization of Alcian Blue staining ( n ) and visualization and quantification of Sox9 expression ( o ) in micromass co-cultures of periosteal cells from Sox9-GFP mice and sorted cell populations from skeletal muscle of CAG-DsRed mice, after 9 days in chondrogenic SD medium (n=4 biologically independent samples). Addition of oleate was used as positive control. Scale bars, 100μm. EC: endothelial cell, MΦ: macrophage. Mean ± s.e.m. One-way ANOVA ( e , f , i , o ), two-way ANOVA ( h , l ) or three-way ANOVA ( g ) with Bonferroni post-hoc test, two-tailed Student’s t-test ( k , m ). For gel source data, see .

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Cell Culture, Injection, Saline, Diffusion-based Assay, Expressing, Positive Control, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a ) Quantification of glucose consumption and lactate secretion (PC, COB: n=6, GCH: n=5 biologically independent samples), glycolytic rate (n=3 biologically independent samples), oxygen consumption (PC, COB: n=7, GCH: n=5 biologically independent samples), glucose oxidation (n=3 biologically independent samples) and palmitate oxidation (n=3 biologically independent samples) in periosteal cells (PC), growth plate-derived chondrocytes (GCH) and calvarial osteoblasts (COB). ( b ) Analysis of adjacent histological sections of a growth plate and fracture callus (PFD7) by Safranin O staining (cartilage) or immunofluorescence for CPT1a or GLUT1 (representative images of 3 mice). Scale bars, 100μm. b: bone, c: cartilage. Dotted white lines delineate cartilage areas. ( c ) Histological visualization and quantification of early chondrogenic (SOX9 + ) and osteogenic (COL1 + ) cells in the callus of fractures (PFD7) transplanted with CAG-DsRed + skeletal stem cells (SSC) transduced with shCPT1a or shSCR (n=3 mice). Scale bars, 50μm. ( d ) Measurement of oxidation of extracellularly added palmitate by periosteal cells in control medium or at different times in SD medium (n=3 biologically independent samples). ( e ) Quantification of FAO-linked OCR in periosteal cells in control medium or at different times in SD medium (3h: n=2, other timepoints: n=3 biologically independent samples). ( f ) Quantification of FAO-linked OCR in periosteal cells, transduced with shSOX9 or shSCR, in control medium or at different times in SD medium (shSCR 12h, shSOX9 control, shSOX9 3h: n=5, all others: n=6 biologically independent samples). ( g ) Quantification of FAO-linked OCR in GCH transduced with shSOX9 or shSCR (n=5 biologically independent samples). ( h ) Quantification of palmitate oxidation in GCH transduced with shSOX9 or shSCR, and in COB transduced with a lentiviral vector encoding SOX9 (SOX9 overexpression; SOX9 OE ) or an empty vector (EV) (n=4 biologically independent samples). Mean ± s.e.m. One-way ANOVA ( a , d , e ) or two-way ANOVA ( f ) with Bonferroni post-hoc test, two-tailed Student’s t-test ( c,g,h ).

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Derivative Assay, Staining, Immunofluorescence, Transduction, Control, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( 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 neighbor embedding (t-SNE) plot of 20,896 non-hematopoietic cells (mixed bone and bone marrow fractions, n=6 mice) based on single cell RNA sequencing 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 sequencing) of FAO- and glycolysis-related genes (rows) in the cells of each cluster (columns). ( d ) qRT-PCR analysis of genes involved in glycolysis ( Glut1 , 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 murine growth plate cartilage and cortical bone biopsies (relative to Actin ). ( 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, 100μm in growth plate images, 50μm in fracture callus images. b: bone, c: cartilage ( 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, 50μm. c: cartilage ( 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 hours of exposure to etomoxir (n=3 biologically independent samples). 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 ).

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Derivative Assay, RNA Sequencing, Expressing, Quantitative RT-PCR, Injection, Immunofluorescence, Staining, Transduction, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a ) Measurement of oxidation of extracellularly added palmitate by periosteal cells in control medium or at different times in LRS medium (n=4 biologically independent samples). ( b ) Quantification of FAO-linked OCR in periosteal cells in control medium or at different times in LRS medium (n=4 biologically independent samples). ( c ) Confocal microscopy of periosteal cells labelled with Red-C12 (fluorescent fatty acid, red) and stained with MitoTracker (mitochondria, green) and DPH (lipid droplets, blue) shows increased co-localization (as quantified by Pearson’s correlation coefficient) of MitoTracker and Red-C12 after exposure of cells for 6 hours to SD (n=4 biologically independent samples). Scale bars, 20μm. ( d ) Immunoblot detection of LC3 in total cell protein extracts of C3H10T1/2 cells and periosteal cells exposed for different times to control or SD medium, with β-actin as loading control. Note increased conversion of LC3-I to LC3-II at early time points, indicative of activation of autophagy (n=2 independent experiments). ( e , f ) Confocal microscopy of C3H10T1/2 cells ( e ; n=3 independent experiments) or periosteal cells ( f ; n=3 biologically independent samples), expressing an RFP-GFP-LC3 tandem construct, shows activation of autophagy with time upon SD, evidenced by increased total number of LC3 puncta per cell and higher percentage of RFP + GFP - puncta. Scale bars, 20μm. ( g ) Confocal microscopy-based visualization (top) and quantification (bottom) of C3H10T1/2 cells, stained with the neutral lipid dye DPH to reveal lipid droplet dynamics at different time points after SD. Cells were transduced with shATG5 to inhibit autophagy or shSCR as a control (n=6 independent experiments). Scale bars, 20μm. ( h ) Quantification of FAO-linked OCR in periosteal cells in control medium or at different times after SD, treated with 10μM chloroquine (CQ) or vehicle (n=3 biologically independent samples). ( i ) Quantification of cell viability of C3H10T1/2 cells and periosteal cells after 72 hours of exposure to control or SD medium in the presence or absence of 50μM (C3H10T1/2 cells) or 10μM (periosteal cells) CQ (n=3 independent experiments for C3H10T1/2 cells, n=3 biologically independent samples for periosteal cells). ( j ) Immunoblot detection of total SOX9 in C3H10T1/2 cells and nuclear SOX9 in periosteal cells exposed for 6 hours (C3H10T1/2 cells) or 24 hours (periosteal cells) to control medium (with DMSO as vehicle control) or medium supplemented with 100μM etomoxir (Eto), with β-actin or Lamin A/C as loading control. ( k ) Cell morphology of growth plate-derived chondrocytes transduced with shSOX9 or shSCR (representative images of 6 biologically independent samples). Scale bar, 100μM. ( l ) qRT-PCR analysis of genes involved in chondrogenesis ( Sox9 , Col2a1 and Acan ) and FAO ( Cpt1a , Acadm and Acadl ) in growth plate-derived chondrocytes transduced with shSOX9 or shSCR (relative to shSCR, n=6 biologically independent samples). Mean ± s.e.m. One-way ANOVA ( a , b , e , f ) or two-way ANOVA ( g , h , i ) with Bonferroni post-hoc test, two-tailed Student’s t-test ( c , l ). For gel source data, see .

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Control, Confocal Microscopy, Staining, Western Blot, Activation Assay, Expressing, Construct, Transduction, Derivative Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a ) Heatmap showing differential expression of cartilage-related genes in C3H10T1/2 cells exposed for different times to SD versus control medium, as determined by mRNA sequencing (n=3 replicates). ( b ) Volcano plot showing significantly enriched and depleted mRNAs in C3H10T1/2 cells exposed for 3 or 6 hours to SD versus control medium, as determined by mRNA sequencing (n=3 replicates). ( c ) Top 10 most significantly enriched transcription factor motifs with normalized enrichment scores (NES) in C3H10T1/2 cells exposed for 3 (left) or 6 (right) hours 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 hours and the Atf4 motif for 6 hours. ( d ) Confocal microscopy of C3H10T1/2 cells stained for FoxO1 after exposure of cells for 3 hours to SD or LRS, in the presence of vehicle (EtOH), oleate (60μM) or PUFA (representative images of 2 independent experiments). Scale bars, 20μm. ( e ) Nuclear FoxO activity in C3H10T1/2 cells exposed for 3 hours to control, SD or LRS medium (n=5 independent experiments). ( f ) Nuclear FoxO activity in skeletal stem cells exposed for 3 hours 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 hours 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 SD, n=3 independent experiments for LRS) and skeletal stem cells (n=3 biologically independent samples) exposed for 24 hours to control, SD or LRS medium supplemented with 1μM 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 hours to control, SD or LRS medium in the presence or absence of doxycycline (dox; 250ng/ml), 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 hours 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 (10nmol) or vehicle (0.2% DMSO in saline) at the fracture site (n=5 mice). Scale bars, 500μm. 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. Mean ± s.e.m. One-way ANOVA ( e , f ) or two-way ANOVA ( g , h , j) with Bonferroni post-hoc test, two-tailed Student’s t-test ( k , l) . For gel source data, see .

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Quantitative Proteomics, Control, Sequencing, Confocal Microscopy, Staining, Activity Assay, Expressing, Transduction, ChIP-qPCR, Western Blot, Saline, Two Tailed Test

Journal: Nature

Article Title: Lipid availability determines skeletal progenitor cell fate via SOX9

doi: 10.1038/s41586-020-2050-1

Figure Lengend Snippet: ( a , b ) Volcano plot showing significantly enriched and depleted mRNAs ( a ) and top 10 most significantly enriched transcription factor motifs with normalized enrichment scores (NES) as determined by i-cisTarget analysis ( b ) in C3H10T1/2 cells exposed for 1 hour to SD versus control medium (n=3 replicates). Motif shown on top is the Forkhead/FoxO motif. ( c ) Confocal microscopy of C3H10T1/2 cells stained for FoxO1 (top) or FoxO3a (bottom) shows increased nuclear localization after exposure of cells for 3 hours to SD or LRS (representative images of 2 independent experiments). Scale bars, 20μm. ( d ) Immunoblot detection of nuclear FoxO1 and FoxO3a in C3H10T1/2 cells exposed for 1, 3 or 6 hours to control or SD medium, with Lamin A/C as loading control (n=2 independent experiments). ( e ) Nuclear FoxO activity in C3H10T1/2 cells exposed for 3 hours to control, SD or LRS medium supplemented with vehicle (EtOH), oleate (60μM) or poly-unsaturated fatty acids (PUFA) (n=3 independent experiments). ( f ) Occupancy of FoxO3a at the Sox9 promoter of Cas9-expressing C3H10T1/2 cells transduced with inducible short guidance RNA against FoxO1 (sgFoxO1), sgFoxO3a or a scrambled sgRNA (sgSCR), exposed for 3 hours to control or SD medium in the presence of doxycycline (250ng/ml), as determined by ChIP-qPCR (n=3 independent experiments). ( g ) Flow cytometric quantification of total SOX9 levels in periosteal cells exposed for 24 hours to control, SD or LRS medium supplemented with 1μM AS1842856 (FoxO inhibitor) or vehicle (DMSO) (n=4 biologically independent samples). ( h ) Histological visualization and quantification of FoxO3a-expressing cells in the central periosteal callus of grafts with or without a filter (0.2μm pore size) at PFD7 (control: n=7, filter 0.2: n=8 mice). Scale bars, 50μm. ( i ) Histological visualization (Safranin O staining) and quantification of cartilage and woven bone in the callus at PFD7 of mice treated daily with AS1842856 (500pmol) or vehicle (0.1% DMSO in saline) at the fracture site (vehicle: n=4, AS1842856: n=5 mice). Scale bars, 500μm. ( j ) Schematic overview of main findings. Mean ± s.e.m. Two-way ANOVA with Bonferroni post-hoc test ( e - g ), two-tailed Student’s t-test ( h , i ). For gel source data, see .

Article Snippet: To silence SOX9, CPT1a, ATG5 or FoxO1/3a we transduced cells, in the presence of 8μg/ml polybrene (Sigma-Aldrich), with a lentivirus carrying a shRNA against SOX9 (Addgene plasmid repository #40645 ; MOI 50), CPT1a (MISSION, Sigma-Aldrich; MOI 25) or ATG5 (MISSION, Sigma-Aldrich; MOI 25), or concomitantly with shRNAs against FoxO1 and FoxO3a (MISSION, Sigma-Aldrich; each at MOI 25).

Techniques: Control, Confocal Microscopy, Staining, Western Blot, Activity Assay, Expressing, Transduction, ChIP-qPCR, Pore Size, Saline, Two Tailed Test