Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Upregulated expression of Sox9 in medial vSMCs of aortic allografts and HMGB1-treated vSMCs (A) Heatmap shows differentially regulated genes (|log2FC(allograft/isograft)| > 1; p adjust <0.01) in the medial cells of non-transplanted aorta from Lewis (n = 4) and BN (n = 4) rats, isografts (Lewis-Lewis. n = 10 rats) and allografts (BN-Lewis. n = 9 rats) transfected with lentiviruses expressing negative control shRNA with a specific tagln promoter (shNC) 2 weeks after transplantation, as detected by preforming RNA sequencing. (B) GO enrichment analysis shows selected GO terms of biological process of differentially regulated genes (DEGs). Size of circles represents the number of DEGs. p adjust values are displayed. (C) Venn diagram shows the overlap between genes in indicated GO terms. (D) qRT-PCR analysis of Sox9 mRNA expression within medial cells of shNC isografts and shNC allografts 2 weeks after transplantation (means ± SEM ∗∗p < 0.01. n = 8 rats per group). (E) Representative cross sections of aortic grafts coimmunostained for Sox9 (red) and Acta2 (green) 2 weeks after transplantation. Cell nuclei were stained with hoechst (blue). White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of Sox9 positive cells in the media of aortic grafts (means ± SEM ∗∗p < 0.01. n = 15 rats per group).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Expressing, Transfection, Negative Control, shRNA, Transplantation Assay, RNA Sequencing Assay, Quantitative RT-PCR, Staining

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Sox9 drives the phenotypic modulation of vSMC following transplantation (A) qRT-PCR analysis of Sox9 , Cnn1 , Acta2 and Tagln mRNA expression within the media of aortic isografts and allograft transfected with sh Sox9 or shNC 2 weeks after transplantation (means ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 8 rats per group). (B) Representative cross sections of non-transplanted aorta, and sh Sox9 -or shNC-transfected aortic grafts immunostained for Cnn1 (green), Acta2 (green) and Tagln (green) 2 weeks after transplantation (Bi). Cell nuclei were stained with hoechst (blue). Red boxes indicate the total sections. White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of Cnn1, Acta2 and Tagln positive area in the media of aortic grafts (Bii) (means ± SEM ∗p < 0.05, ∗∗∗p < 0.001. n = 15 rats per group). (C and D) qRT-PCR and western blotting analyses of Myocd , Srf , Sox9 , Cnn1 , Acta2 and Tagln expression in cultured vSMCs transfected with lentiviruses carring Sox9 -targeting shRNA (sh Sox9 ) with a specific Tagln promoter or negative control shRNA (shNC) followed by HMGB1 (100 ng/mL) stimulation or not (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Transplantation Assay, Quantitative RT-PCR, Expressing, Transfection, Staining, Western Blot, Cell Culture, shRNA, Negative Control

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Sox9 facilitates vSMC proliferation, migration and transplant arteriosclerosis in rat aortic allografts (A) Representative cross sections of shNC- or sh Sox9 -transfected isografts and allografts immunostained for PCNA 2 weeks after transplantation. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of PCNA-positive cells in the media of aortic grafts (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 15 rats per group). (B and C) vSMCs transfected with sh Sox9 or negative control shNC were stimulated with or without HMGB1 (100 ng/mL), cell proliferation was evaluated by CCK-8 and BrdU incorporation assays as the measured absorbance at 450 nm (means ± SEM ∗p < 0.05. n = 5). (D) Representative images of migrated vSMCs on the bottom of transwell membrane as detected by Transwell assays (means ± SEM ∗p < 0.05. n = 5). (E) Representative images to show H&E staining, EVG staining and immunofluorescence staining of vSMC marker Acta2 in cross sections of aortic isografts and allografts transfected with shNC or sh Sox9 8 weeks after aortic transplantation. Green arrows denote the internal elastic lamina. White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. (F–H) Quantification of of intimal area, intima/media ratio and lumen stenosis ratio (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 18 rats per group).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Migration, Transfection, Transplantation Assay, Negative Control, CCK-8 Assay, BrdU Incorporation Assay, Staining, Immunofluorescence, Marker

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Autophagy is associated with increased Sox9 expression in vSMCs (A) Representative cross sections of non-transplanted aorta and shNC-transfected aortic grafts coimmunostained for LC3Ⅰ/Ⅱ (red) and Acta2 (green) 2 weeks after transplantation. Cell nuclei were stained with hoechst (blue). White dotted line outlines the media of the vascular wall. A, adventitial; L, lumen. Scale bar: 100 μm. Quantification of LC3Ⅰ/Ⅱ-positive area in the media of aortic grafts (means ± SEM ∗∗∗p < 0.001. n = 15 rats per group). (B) Transmission electron microscopy images of double membrane autophagosome and single membrane autophagolysosome structures in aortic isografts and allografts transfected with shNC. Red boxes show the enlarged sections of aortic grafts. (C) LC3Ⅰ/Ⅱ-stained fluorescent microscopy images of vSMCs treated with HMGB1 (100 ng/mL) for 48 h, transmission electron microscopy images of autophagosomes or autophagolysosomes in vSMCs stimulated with HMGB1 (100 ng/mL). Red boxes show the enlarged sections. (D) vSMCs were transfected with small interfering RNA targeting Atg5 (si Atg5 ) or its corresponding negative control (siNC) followed by HMGB1 (100 ng/mL) stimulation, Sox9 mRNA expression was measured by qRT-PCR (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (E) Western blotting analysis of Sox9, Atg5, p62 and LC3Ⅰ/Ⅱ expression in vSMCs transfected with si Atg5 or siNC followed by HMGB1 (100 ng/mL) stimulation (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (F and G) vSMCs pretreated with or without Bafilomycin A1 (200 nM) were stimulated with HMGB1. qRT-PCR analysis of Sox9 mRNA expression. n = 4. Western blotting analysis of Sox9, p62 and LC3Ⅰ/Ⅱ protein expression (means ± SEM ∗p < 0.05. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Expressing, Transfection, Transplantation Assay, Staining, Transmission Assay, Electron Microscopy, Microscopy, Small Interfering RNA, Negative Control, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Autophagy promotes vSMC phentopic modulation by upregulating Sox9 (A and B) qRT-PCR and western blotting analyses of Cnn1 , Acta2 and Tagln mRNA and protein expression in vSMCs transfected with si Atg5 or siNC followed by HMGB1 stimulation (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (C and D) vSMCs pretreated with or without bafilomycin A1 were stimulated with HMGB1, mRNA and protein expression of Cnn1 , Acta2 and Tagln were measured by qRT-PCR and western blotting (means ± SEM ∗p < 0.05. n = 4). (E and F) qRT-PCR and western blotting analyses of Sox9 , Cnn1 , Acta2 and Tagln expression in vSMCs transfected with plasmids overexpressing Sox9 ( Sox9 oe) or its control vector (vector) (means ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 4). (G) vSMCs were cotransfected with Atg5 siRNA and Sox9 overexpressing plasmid together with HMGB1 (100 ng/mL) stimulation, protein expression of Sox9, Cnn1, Acta2 and Tagln were determined (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (H and I) BrdU incorporation assay was performed in si Atg5 -transfected and Bafilomycin A1-pretreated vSMCs exposed to HMGB1 (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 3). (J and K) The migration ability of si Atg5 -transfected and Bafilomycin A1-pretreated vSMCs exposed to HMGB1 was evaluated by Transwell assay (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 3).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, BrdU Incorporation Assay, Migration, Transwell Assay

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: Autophagy-mediated Sox9 expression relies on p27 degradation (A and B) qRT-PCR and western blotting analyses of p27 Kip1 ( p27 ) expression in vSMCs exposed to HMGB1 (100 ng/mL) for indicated time (means ± SEM ∗p < 0.05. n = 4). (C and D) vSMCs were stimulated with HMGB1 for 48 h, co-IP assay was performed to analyze the ubiquitination of p27 using anti-ubiquitin (n = 4); Binding of p27 and p62 was determined by immunoprecipitating p27 (n = 4). (E) vSMCs pretreated with Bafilomycin A1 (200 nM) or MG132 (5 μM) were stimulated with or without HMGB1 (100 ng/mL) for 48 h, p27 protein level was determined by western blotting analysis, and densitometric analysis of blots was performed (means ± SEM ∗p < 0.05. n = 4). (F) western blotting analysis and quantification of p27 expression in vSMCs transfected with si Atg5 or siNC followed by HMGB1 stimulation (means ± SEM ∗p < 0.05. n = 4). (G and H) qRT-PCR and western blotting analyses of p27 expression in vSMCs transfected with si p27 or siNC (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (I and J) vSMCs were cotransfected with sh Sox9 and si p27 , Cnn1, Acta2 and Tagln expression were detected by western blotting (means ± SEM ∗p < 0.05. n = 4); Cell proliferation was evaluated by BrdU incorporation assay (means ± SEM ∗p < 0.05. n = 3); Cell migration was detected by Transwell assay (means ± SEM ∗p < 0.05. n = 3). (L and M) vSMCs were cotransfected with si Atg5 and si p27 together with or without HMGB1, mRNA expression of p27 and Sox9 were detected by qRT-PCR (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 3); p27 and Sox9 protein expression were measured by western blotting (means ± SEM ∗p < 0.05. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Transfection, BrdU Incorporation Assay, Migration, Transwell Assay

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet: p27 associates with p130/E2F4 complex on Sox9 promoters (A) vSMCs were cotransfected with pGL3- Sox9 reporter construct and p27 overexpressing plasmid or empty vector for 48 h, Sox9 promoter activity was measured by dual luciferase assays (means ± SEM ∗p < 0.05. n = 3). (B) Dual luciferase assays were performed to evaluate Sox9 promoter activity in vSMCs cotransfected with pGL3- Sox9 reporter construct and si p27 for 48 h (means ± SEM ∗p < 0.05. n = 3). (C) Confirmation of the binding of p27 to Sox9 promoter in vSMCs by ChIP assay followed by qRT-PCR and agarose gel electrophoresis. An anti-IgG antibody was used as the negative control, an anti-Histone H3 antibody was used as the positive control and no antibody was added as the empty control (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 4). (D) Coimmunoprecipitation (co-IP) of p27 and p130/E2f4 complex in vSMCs (n = 4). (E) ChIP of p130 and E2F4 protein in Sox9 promoter in vSMCs (means ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001. n = 4). (F) vSMCs were cotransfected with pGL3- Sox9 reporter construct and p27 overexpressing plasmid or p130 siRNA or E2F4 siRNA, Sox9 promoter activity was measured by dual luciferase assays (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4). (G and H) qRT-PCR and western blotting analyses of Sox9 , Cnn1 , Acta2 and Tagln expression in vSMCs transfected with si p130 , si E2F4 or siNC for 48 h (means ± SEM ∗p < 0.05, ∗∗p < 0.01. n = 4).

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Construct, Plasmid Preparation, Activity Assay, Luciferase, Binding Assay, Quantitative RT-PCR, Agarose Gel Electrophoresis, Negative Control, Positive Control, Co-Immunoprecipitation Assay, Western Blot, Expressing, Transfection

Journal: iScience

Article Title: Sox9 mediates autophagy-dependent vascular smooth muscle cell phenotypic modulation and transplant arteriosclerosis

doi: 10.1016/j.isci.2022.105161

Figure Lengend Snippet:

Article Snippet: Rabbit anti-Sox9 antibody , Abcam , Cat#ab185230; RRID: AB_2715497.

Techniques: Recombinant, CCK-8 Assay, Labeling, Sonication, Chromatin Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Sequencing, Plasmid Preparation, Software, Imaging

Journal: Journal of visualized experiments : JoVE

Article Title: Generation of Organoids from Mouse Extrahepatic Bile Ducts

doi: 10.3791/59544

Figure Lengend Snippet: Materials

Article Snippet: Prepare seeding medium and washing buffer ( ) in 50 mL conical tubes and keep them at 4 °C or on ice until use. table ft1 table-wrap mode="anchored" t5 Name Company Catalog Number Comments L-WRN cell culture medium Advanced DMEM/F12 Life Technologies 12634–010 Fetal Bovine Serum (FBS) 1% Life Technologies 10437–028 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 Washing buffer Phosphate Buffered Saline (PBS) 50 mL Life Technologies 10010–023 Penicillin-Streptomycin 125 U/mL Life Technologies 15140–122 Amphotericin B 6.25 μg/mL Life Technologies 15290–018 Organoid culture medium L-WRN Conditioned medium 1:1 ATCC CRL-3276 Advanced DMEM/F12 1:1 Life Technologies 12634–010 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 N-Glutamine 10 μl/mL Life Technologies 35050–061 N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES 10 mM Life Technologies 15630–080 B27 10 μl/mL Gibco 17504–044 N2 10 μl/mL Gibco 17502–048 Organoid seeding medium Organoid culture medium Epidermal growth factor (EGF) 50 ng/mL Invitrogen PMG8041 Fibroblast growth factor-10 (FGF10) 100 ng/mL PeproTech 100–26 Primary antibodies Anti-Cytokeratin 19 (CK19) antibody, Rabbit 1:250 Abcam ab53119 Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit 1:200 Santa Cruz sc-20095 Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit 1:2000 DSRB F109-D12 E-cadherin antibody, Goat 1:500 Santa Cruz sc-31020 Acetylated α-tubulin antibody, Mouse 1:500 Sigma-Aldrich T6793 Secondary antibodies 488 labeled anti-rabbit, Donkey IgG 1:1000 Invitrogen A-21206 594 labeled anti-goat, Donkey IgG 1:1000 Invitrogen A-11058 568 labeled anti-mouse, Goat IgG2b 1:500 Invitrogen A-21144 TopFlash Wnt reporter assay TopFlash HEK293 cell line ATCC CRL-1573 Luciferase Assay Kit Biotium 30003–2 0.05% Trypsin-EDTA Life Technologies 25300054 0.4% Trypan Blue Solution Life Technologies 15250061 Additional materials and reagents Basement matrix, phenol free Matrigel CORNING 356237 Dissociation buffer, Accutase Gibco A1110501 Cell culture freezing medium, Recovery Life Technologies 12648010 Cell strainer (70 μm, steriled) Fisherbrand 22363548 Guanidinium thiocyanate-phenol RNA extraction, TRIzol Invitrogen 15596026 Specimen processing gel, HistoGel Thermo Fisher Scientific HG-4000–012 Universal mycoplasma detection kit ATCC 30–1012K 1.5 mL microcentrifuge tube Fisherbrand 05–408–129 24 well plate USA Scientific CC7682–7524 50 mL conical centrifuge tube Fisher scientific 14–432–22 Fluorescence microscope Nikon Eclipse E800 Inverted microscope Biotium 30003–2 Necropsy tray Fisherbrand 13–814–61 Open in a separate window Materials Set up a surgical table ( ).

Techniques: Cell Culture, Saline, Labeling, Reporter Assay, Luciferase, RNA Extraction, Fluorescence, Microscopy, Inverted Microscopy

Journal: Journal of visualized experiments : JoVE

Article Title: Generation of Organoids from Mouse Extrahepatic Bile Ducts

doi: 10.3791/59544

Figure Lengend Snippet: (A-C). EHBDOs were analyzed by immunofluorescence staining for markers epithelial (A, B. E-cadherin, red), progenitor (A. PDX1, green), and differentiated (B. CK19, green; and C. a-AT, red) biliary cells. Scale bars = 25 μm. *, lumen. (D). EHBDOs were analyzed for abundance of Pdx1, Ck19, Sox9, Aqp1, and Cftr mRNA by qRT-PCR (mean +/− SEM relative to expression of Hprt).

Article Snippet: Prepare seeding medium and washing buffer ( ) in 50 mL conical tubes and keep them at 4 °C or on ice until use. table ft1 table-wrap mode="anchored" t5 Name Company Catalog Number Comments L-WRN cell culture medium Advanced DMEM/F12 Life Technologies 12634–010 Fetal Bovine Serum (FBS) 1% Life Technologies 10437–028 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 Washing buffer Phosphate Buffered Saline (PBS) 50 mL Life Technologies 10010–023 Penicillin-Streptomycin 125 U/mL Life Technologies 15140–122 Amphotericin B 6.25 μg/mL Life Technologies 15290–018 Organoid culture medium L-WRN Conditioned medium 1:1 ATCC CRL-3276 Advanced DMEM/F12 1:1 Life Technologies 12634–010 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 N-Glutamine 10 μl/mL Life Technologies 35050–061 N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES 10 mM Life Technologies 15630–080 B27 10 μl/mL Gibco 17504–044 N2 10 μl/mL Gibco 17502–048 Organoid seeding medium Organoid culture medium Epidermal growth factor (EGF) 50 ng/mL Invitrogen PMG8041 Fibroblast growth factor-10 (FGF10) 100 ng/mL PeproTech 100–26 Primary antibodies Anti-Cytokeratin 19 (CK19) antibody, Rabbit 1:250 Abcam ab53119 Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit 1:200 Santa Cruz sc-20095 Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit 1:2000 DSRB F109-D12 E-cadherin antibody, Goat 1:500 Santa Cruz sc-31020 Acetylated α-tubulin antibody, Mouse 1:500 Sigma-Aldrich T6793 Secondary antibodies 488 labeled anti-rabbit, Donkey IgG 1:1000 Invitrogen A-21206 594 labeled anti-goat, Donkey IgG 1:1000 Invitrogen A-11058 568 labeled anti-mouse, Goat IgG2b 1:500 Invitrogen A-21144 TopFlash Wnt reporter assay TopFlash HEK293 cell line ATCC CRL-1573 Luciferase Assay Kit Biotium 30003–2 0.05% Trypsin-EDTA Life Technologies 25300054 0.4% Trypan Blue Solution Life Technologies 15250061 Additional materials and reagents Basement matrix, phenol free Matrigel CORNING 356237 Dissociation buffer, Accutase Gibco A1110501 Cell culture freezing medium, Recovery Life Technologies 12648010 Cell strainer (70 μm, steriled) Fisherbrand 22363548 Guanidinium thiocyanate-phenol RNA extraction, TRIzol Invitrogen 15596026 Specimen processing gel, HistoGel Thermo Fisher Scientific HG-4000–012 Universal mycoplasma detection kit ATCC 30–1012K 1.5 mL microcentrifuge tube Fisherbrand 05–408–129 24 well plate USA Scientific CC7682–7524 50 mL conical centrifuge tube Fisher scientific 14–432–22 Fluorescence microscope Nikon Eclipse E800 Inverted microscope Biotium 30003–2 Necropsy tray Fisherbrand 13–814–61 Open in a separate window Materials Set up a surgical table ( ).

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: Journal of visualized experiments : JoVE

Article Title: Generation of Organoids from Mouse Extrahepatic Bile Ducts

doi: 10.3791/59544

Figure Lengend Snippet: Primers.

Article Snippet: Prepare seeding medium and washing buffer ( ) in 50 mL conical tubes and keep them at 4 °C or on ice until use. table ft1 table-wrap mode="anchored" t5 Name Company Catalog Number Comments L-WRN cell culture medium Advanced DMEM/F12 Life Technologies 12634–010 Fetal Bovine Serum (FBS) 1% Life Technologies 10437–028 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 Washing buffer Phosphate Buffered Saline (PBS) 50 mL Life Technologies 10010–023 Penicillin-Streptomycin 125 U/mL Life Technologies 15140–122 Amphotericin B 6.25 μg/mL Life Technologies 15290–018 Organoid culture medium L-WRN Conditioned medium 1:1 ATCC CRL-3276 Advanced DMEM/F12 1:1 Life Technologies 12634–010 Penicillin-Streptomycin 100 U/mL Life Technologies 15140–122 N-Glutamine 10 μl/mL Life Technologies 35050–061 N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES 10 mM Life Technologies 15630–080 B27 10 μl/mL Gibco 17504–044 N2 10 μl/mL Gibco 17502–048 Organoid seeding medium Organoid culture medium Epidermal growth factor (EGF) 50 ng/mL Invitrogen PMG8041 Fibroblast growth factor-10 (FGF10) 100 ng/mL PeproTech 100–26 Primary antibodies Anti-Cytokeratin 19 (CK19) antibody, Rabbit 1:250 Abcam ab53119 Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit 1:200 Santa Cruz sc-20095 Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit 1:2000 DSRB F109-D12 E-cadherin antibody, Goat 1:500 Santa Cruz sc-31020 Acetylated α-tubulin antibody, Mouse 1:500 Sigma-Aldrich T6793 Secondary antibodies 488 labeled anti-rabbit, Donkey IgG 1:1000 Invitrogen A-21206 594 labeled anti-goat, Donkey IgG 1:1000 Invitrogen A-11058 568 labeled anti-mouse, Goat IgG2b 1:500 Invitrogen A-21144 TopFlash Wnt reporter assay TopFlash HEK293 cell line ATCC CRL-1573 Luciferase Assay Kit Biotium 30003–2 0.05% Trypsin-EDTA Life Technologies 25300054 0.4% Trypan Blue Solution Life Technologies 15250061 Additional materials and reagents Basement matrix, phenol free Matrigel CORNING 356237 Dissociation buffer, Accutase Gibco A1110501 Cell culture freezing medium, Recovery Life Technologies 12648010 Cell strainer (70 μm, steriled) Fisherbrand 22363548 Guanidinium thiocyanate-phenol RNA extraction, TRIzol Invitrogen 15596026 Specimen processing gel, HistoGel Thermo Fisher Scientific HG-4000–012 Universal mycoplasma detection kit ATCC 30–1012K 1.5 mL microcentrifuge tube Fisherbrand 05–408–129 24 well plate USA Scientific CC7682–7524 50 mL conical centrifuge tube Fisher scientific 14–432–22 Fluorescence microscope Nikon Eclipse E800 Inverted microscope Biotium 30003–2 Necropsy tray Fisherbrand 13–814–61 Open in a separate window Materials Set up a surgical table ( ).

Techniques: Sequencing

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) Analysis of side population (SP) in human lung carcinoma H460 cells in the presence or absence of fumitremorgin C (FTC) using FACS. SP cells ( box) were determined by their disappearance in the presence of FTC and were shown as percentage of the pool population. CSCs were isolated based on SP phenotype and their aggressive features were validated in vitro and in vivo as shown in . ( b ) Analysis of EMT markers and ABCG2 transporter in human normal lung epithelial BEAS-2B (BC) cells and SP (CSC) and NSP (non-CSC) H460 cells using Western blotting. Immunoblot signals from three-independent experiments (one of which is shown here) were quantified by densitometry, revealing a dominant overexpression of SLUG in SP cells. ( c ) Western blot analysis of SLUG, VIM and CDH1 in SLUG knockdown (shSNAI2) and control (shCON) H460 cells. ( d ) Protein expression of SLUG and SOX9 in clinical lung cancer and matched normal lung tissues. Blots were reprobed with anti-β-actin (ACTB) antibody to confirm equal loading of the samples. Quantitative analysis of SLUG and SOX9 levels revealed a striking difference between normal (N) and tumor (T) tissues at the significance level in two-sided Student’s t -test of P < 0.03 and P < 0.003, respectively. ( e and f ) SLUG and SOX9 knockdown and overexpression experiments were performed using H460 cells treated with lentiviral particles carrying shSNAI2, shSOX9 or shCON and nucleofection of GFP, SNAI2 or SOX9 overexpression plasmids, as described under “Materials and methods”. Analysis of ( e ) tumor sphere formation and ( f ) SP in various clones of H460 cells. Scale bar = 200 µm. Data are mean ± S.D. (n = 4). * P < 0.05 vs . shCON cells; two-sided Student’s t -test.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Isolation, In Vitro, In Vivo, Western Blot, Over Expression, Knockdown, Control, Expressing, Clone Assay

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) Immunohistochemistry analysis of SLUG and SOX9 in isolated lungs from mice bearing shSNAI2, shSOX9 and shCON cells. Scale bar = 200 µm. ( b ) Correlation analysis of the protein expression of SLUG and SOX9 in clinical lung tumor ( red triangle ) and matched normal ( blue square ) samples. ( c ) Western blot analysis of SLUG and SOX9 expression in shSNAI2, shSOX9 and shCon H460 and A549 cells. ( d ) shSNAI2 and shCON cell lysates were prepared, immunoprecipitated with control IgG or anti-SOX9 antibody, and probed with anti-SLUG antibody. Immunoblots were performed on cell lysates used as input for immunoprecipitation (IP) using anti-β-actin (ACTB) antibody to confirm equal loading of the samples. ( e ) Chromatin IP (ChIP) analysis of SLUG binding on SOX9 promoter. Sheared chromatin was immunoprecipitated using ChIP-grade anti-SLUG antibody or IgG control, and ChIP DNA was quantified by real-time PCR with primers specific to human SOX9 promoter (see for primer sequences). Data are mean ± S.D. (n = 3). ( f ) SLUG overexpressing or knockdown H460 cells were seeded onto type I collagen-coated slides. The cells were immunostained for SLUG ( red ), SOX9 ( blue ) and examined under a confocal fluorescence microscope. Colocalization of SLUG and SOX9 is shown in the merged display ( purple ). Images were taken pairwise with the same instrumental setting. Scale bar = 50 µm. ( g ) In-cell co-IP using proximity ligation (Duolink ® ) assay as described under “Materials and methods”. Cells were similarly seeded onto type I collagen-coated slides and in-cell SLUG-SOX9 interaction ( red ) was determined using the Duolink ® assay. Scale bar = 50 µm.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Immunohistochemistry, Isolation, Expressing, Western Blot, Immunoprecipitation, Control, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Knockdown, Fluorescence, Microscopy, Co-Immunoprecipitation Assay, Ligation

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) H460 cells were treated with proteasome inhibitor MG132 (25–100 µM) for 24 hours and SOX9 expression was determined by Western blotting. ( b ) Quantitative real-time PCR of SOX9 mRNA expression in SLUG overexpressing or knockdown H460 cells. ( c ) H460 cells were treated with MG132 (50 µM) to prevent proteasomal degradation of SOX9 and cell lysates were prepared and immunoprecipitated using control IgG or anti-SOX9 antibody. The immune complexes were analyzed for ubiquitin at various times (0–3 hours) by Western blotting. Immunoblots were performed on cell lysates used as IP input using anti-β-actin (ACTB) antibody to confirm equal loading of the samples. ( d ) Analysis of SOX9 ubiquitination (Poly Ub SOX9) in shSNAI2 and shCON H460 and A549 cells in the presence or absence of MG132 at 3 hours, in which ubiquitination was found to be maximal. Equal amounts of protein were loaded in each lane. Data are mean ± S.D. (n = 3). * P < 0.05 vs . shCON cells in the presence of MG132; two-sided Student’s t -test. ( e ) Various clones of H460 cells, including shCON, shSNAI2, shCON/SNAI2, shSNAI2/SNAI2, shCON/SOX9 and shSNAI2/SOX9 cells were treated with protein translation inhibitor cycloheximide (CHX; 10 µg/mL) for various times to follow the degradation of SOX9 protein. Cell lysates were prepared and SOX9 expression was determined by Western blotting. Representative immunoblots of SOX9 and loading control β-actin (ACTB) are shown (see also ). SOX9 expression time profile was plotted and SOX9 half-life was calculated and shown. Data are mean ± S.D (n = 3). * P < 0.05 vs . shCON cells; two-sided Student’s t -test. No significant differences were observed when compared between shSNAI2 and shSNAI2/SOX9 cells.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Knockdown, Immunoprecipitation, Control, Ubiquitin Proteomics, Clone Assay

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: ( a ) LUC2-labeled shCON or shSNAI2 H460 cells were transfected with SOX9 or GFP control plasmid and were analyzed for tumor sphere formation. Scale bar = 300 µm. ( b,c ) LUC2-labeled shCON or shSNAI2 H460 cells were transfected with SOX9 or GFP plasmid and injected into NSG mice via tail vein at the dose of 1×10 6 cells/mouse. ( b ) Representative bioluminescence of mice taken at the time of inoculation (week 0) and at 2 and 3 weeks post-injection ( top ) and H&E micrographs of lung tissues ( bottom ). Scale bar = 200 µm. ( c ) Normalization of tumor signals at various times to their initial signal at week 0 and relative to shCON/GFP or shSNAI2/GFP cells. Data are mean ± S.D. (n = 3 or 4). * P < 0.05 vs . shCON/GFP or shSNAI2/GFP cells; two-sided Student’s t -test.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Labeling, Transfection, Control, Plasmid Preparation, Injection

Journal: Oncogene

Article Title: SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma

doi: 10.1038/onc.2015.351

Figure Lengend Snippet: In metastatic tumor cells, increased SLUG expression stabilizes SOX9 through their binding interaction, which inhibits SOX9 ubiquitination and proteasomal degradation. SOX9 stabilization promotes the expansion of CSCs and subsequent cancer metastasis.

Article Snippet: Lentiviral plasmids carrying shRNA sequence against human SOX9 were obtained from Addgene (Cambridge, MA; plasmid 40644) and Santa Cruz Biotechnology (Dallas, TX; sc-36533-SH) and shSOX9 lentivirus production was performed using HEK293T packaging cells (ATCC) in conjugation with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene, plasmids 8454 and 8455).

Techniques: Expressing, Binding Assay, Ubiquitin Proteomics

Journal: Oncogene

Article Title: Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis

doi: 10.1038/s41388-020-01564-w

Figure Lengend Snippet: a Box plot representing the expression of vital cancer stem cell markers (PROM1, SOX9, EPCAM, and CD44) and self-renewal genes on PDAC patients (N=179) versus normal controls (N=171, * P<0.05). b Bar graph showing quantitative analysis of CSC markers (SOX9, CD44, EpCAM, and CD133) expression in a stained tissue microarray (Normal =3, Grade 2= 6, and Grade 3 =6). Mean fluorescent intensities for Red, Green, and Blue (DAPI) stains were noted for each image. Red and green staining was then normalized by blue stain to get normalized mean fluorescent intensities ( * P<0.05, ** P<0.01, *** P<0.001). c Representative immunofluorescence images of human tissue microarray with pancreatic cancer and normal pancreatic cores stained with SOX9, CD44, EpCAM, and CD133. CSC and self-renewal markers expression in normal pancreatic and PDAC pathological stage II, tumor grade 2 malignant, PDAC pathological stage III, tumor grade 3 malignant, tissues. d Bar graph depicting relative expression of SOX9 between gemcitabine sensitive and resistant PDAC cell lines. e Dot plot demonstrating IHC quantification of Sox 9 expression in a subset of patients treated with chemotherapy (N=7) and no chemotherapy (N=4, * P<0.05). f Representative images were showing SOX9 protein expression in chemotherapy-treated and un-treated human PDAC tissues.

Article Snippet: SOX9 human siRNA oligo duplex (cat#: SR304532) was purchased from Origene Technologies.

Techniques: Expressing, Staining, Microarray, Immunofluorescence

Journal: Oncogene

Article Title: Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis

doi: 10.1038/s41388-020-01564-w

Figure Lengend Snippet: a NSP and SP cells were isolated from SW1990 PC cells and subjected to drug treatment for 24 h. Immunoblot analysis of CSC (ALDH1, CD44v6, SOX2, ESA, and ABCG2), oncogenic/proliferative (total and activated ERK) and migratory (total and activated FAK) effectors upon afatinib treatment. ( b - e ) Co-immunolocalization analysis of CSC proteins in KPC tumoroids and PC xenograft tissues. Bar graph demonstrating quantification of mean fluorescent intensities of CSC proteins (pEGFR, CD44v6, ESA, ALDH1, and CD133) in KPC organoids ( b ) and PC xenograft tissues (N=5/group, * P<0.05, ** P<0.01, *** P<0.001) ( d ). Representative confocal microscopic images are showing a response of CSC markers upon afatinib/gemcitabine treatment in KPC tumoroids (c) and xenograft tissues ( e ), scale bars=20 μM. ( f ) Western blot analysis of CSC markers (SOX9, PAF1/PD2, and NANOG) in mouse UN-KPC Y846 PC cells treated with/without afatinib and gemcitabine and combination. Beta-actin served as loading control in NSP-SW1990, SP-SW1990, and UN-KPC Y846 PC cells.

Article Snippet: SOX9 human siRNA oligo duplex (cat#: SR304532) was purchased from Origene Technologies.

Techniques: Isolation, Western Blot, Control

Journal: Oncogene

Article Title: Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis

doi: 10.1038/s41388-020-01564-w

Figure Lengend Snippet: a PCR array for CSC and self-renewal markers in SP/CSC cells (SP-SW1990) after 4 days of treatment with afatinib. b RT-PCR analysis showing downregulation of FOXA2 and SOX9 mRNA (Top 3 images) and immunoblot analysis of FOXA2 and β-actin (loading control) proteins (bottom 2 blots) in PCSC in response to afatinib treatment. c Transient suppression of EGFR in SP-SW1990 cells and its effects on CSC markers (FOXA2, SOX9, DCAMLK1, NANOG, and OCT4) and oncogenic signaling molecules (pERK) along with a reduction of total EGFR protein. d Immunoblot analysis of PCSC cells transiently knockdown for FOXA2 and its impact on SOX9 protein. e Immunoblot analysis showing a reduction in FOXA2 and SOX9 protein expression upon dose-dependent (IC 20 and IC 50 concentrations) treatments with afatinib alone in SPPC cells. f Immunofluorescence analysis of FOXA2 and SOX9 protein co-expression in afatinib-treated KPC tumoroids and mouse primary xenograft tumors. g Immunoblot analysis of FOXA2 and SOX9 protein expression in PC cells treated with afatinib/gemcitabine alone and combination therapies. h Schematic model of the hypothesized signaling axis by which afatinib inhibits the PCSC, scale bars= 5 μM and 2 μM.

Article Snippet: SOX9 human siRNA oligo duplex (cat#: SR304532) was purchased from Origene Technologies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Knockdown, Expressing, Immunofluorescence

Journal: Scientific Data

Article Title: A single-cell transcriptomic dataset of pluripotent stem cell-derived astrocytes via NFIB / SOX9 overexpression

doi: 10.1038/s41597-024-03823-x

Figure Lengend Snippet: Schematic overview of the experimental design. ( a ) Generation of astrocytes from induced pluripotent stem cells (iPSCs). Ubiq, ubiquitin; rtTA, reverse tetracycline-controlled transactivator; Dox, doxycycline; Puro, puromycin (for cell selection). Day 0 is the starting time point of Dox induction. ( b ) Workflow of single-cell RNA sequencing. To establish the astrocyte differentiation path, cells derived from the Monoclonal iPSC1 line were collected on Day 0, 1, 3, 8, 14, and 21. To characterize the molecular signatures of induced astrocytes, astrocytes derived from the iPSC1 and iPSC2 lines on Day 21 were collected. iPSC1: DYR0100 cell line; iPSC2: BIONi037-A cell line; Monoclonal iPSC1: a single colony picked from iPSC1. ( c ) Bioinformatics pipeline for sequencing data analysis. Raw sequencing data were pre-processed by Cell Ranger and Seurat. Plots were generated using R. This figure was created using BioRender ( https://biorender.com/ ).

Article Snippet: Similarly, the full-length cDNA of the mouse Sox9 gene followed by the puromycin selection gene was amplified from the tetO- Sox9 -Puro plasmid (Addgene #117269) to create Sox9 -T2A-Puro.

Techniques: Ubiquitin Proteomics, Selection, RNA Sequencing, Derivative Assay, Sequencing, Generated

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