gls1  (Proteintech)

Journal: The Journal of Clinical Investigation

Article Title: α -Ketoglutarate protects against cartilage damage via epigenetically driven metabolic reprogramming in osteoarthritis models

doi: 10.1172/JCI172380

Figure Lengend Snippet: ( A ) Heatmap of amino acid content analyzed by LC-MS in sham- and DMM-operated knee cartilage ( n = 3 and 2, respectively) of mice at 8 weeks after surgery. ( B ) Heatmap of amino acid levels analyzed by LC-MS in chondrocytes treated with or without IL-1β (5 ng/mL) for 36 hours ( n = 4 per group). Ctrl, control. ( C ) Intracellular Gln levels in IL-1β–treated chondrocytes supplemented with Gln (4 or 8 mM) for 24 hours ( n = 3 per group). ( D and E ) Quantitative analysis of qRT-PCR ( n = 4 per group) ( D ) and Western blot ( E ) analysis for the indicated anabolic and catabolic factors in IL-1β–treated chondrocytes supplemented with Gln (4 or 8 mM) for 24 hours. ( F ) Schematic of Gln metabolism. ( G ) Western blot analysis for SLC1A5 and GLS1 in chondrocytes treated with or without IL-1β for 24 hours ( n = 3 per group). ( H ) Representative images of SLC1A5 and GLS1 immunofluorescence and mean intensity in joint cartilage of sham- or DMM-operated mice at 8 weeks after surgery ( n = 5 mice per group). Scale bars: 20 μm. ( I ) Representative images of SLC1A5 and GLS1 immunofluorescence and mean intensity in mice fed a standard diet (SD) and those fed an HFD ( n = 5 mice per group). Scale bars: 20 μm. ( J ) Representative images from safranin O staining and SLC1A5 and GLS1 immunofluorescence in undamaged ( n = 10), mildly damaged ( n = 10), and severely damaged (SevD) ( n = 7) human OA cartilage. Scale bars: 20 μm. ( K ) Gln and Glu levels in undamaged (UD) ( n = 10), mildly damaged (MD) ( n = 10) and SevD ( n = 7) human OA cartilage. The correlation between Gln levels and ICRS grading scores is shown in the right column. Scale bars: 200 μm. The data are presented as box plots or as the mean ± SEM, and the dots represent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against SOX9 (Abcam, ab3697; 1:1,000); Collagen II (Abcam, ab85266; 1:1,000); MMP3 (Abcam, ab52915; 1:1,000); MMP13 (Proteintech, 18165-1-AP; 1:1,000); ADAMTS5 (Abcam, ab41037; 1:200); NOS2 (Abcam, ab178945; 1:1,000); SLC1A5 (Sigma, HPA035239; 1:1,000); GLS1 (Proteintech; 12855-1-ap; 1:1,000); GS (Abcam, ab176562; 1:1,000); NF-κB p65 (Santa Cruz Biotechnology; sc-8008; 1:1,000); lamin A/C (Santa Cruz Biotechnology; sc-6215; 1:5,000); phospho-IKKα/β (Cell Signaling Technology; 2694P; 1:1,000); IKKα/β (Abcam, ab178870; 1:1,000); phospho-IκBα (Affinity, AF2002; 1:1,000); IκBα (Affinity, AF5002; 1:1,000); TRAF6 (Santa Cruz Biotechnology; sc-8409; 1:200); K63 Ub (Cell Signaling Technology; 5621S; 1:1,000); and GAPDH (Abcam, ab8245; 1:2,000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: The Journal of Clinical Investigation

Article Title: α -Ketoglutarate protects against cartilage damage via epigenetically driven metabolic reprogramming in osteoarthritis models

doi: 10.1172/JCI172380

Figure Lengend Snippet: ( A and B ) mRNA ( A ) and Western blot ( B ) analysis of the indicated anabolic and catabolic factors in chondrocytes treated with BPTES or IL-1β alone or with combined BPTES and IL-1β for 24 hours. The culture medium contained 4 mM Gln. Blots are representative of 3 independent experiments. Ctrl, control. ( C ) Representative safranin O/fast green staining images and OARSI scores ( n = 8 mice per group) in Gls1 −/− mice and their Gls1 fl/fl control littermates at 8 weeks after sham and DMM surgery. ( D ) Representative MMP13 and NOS2 IHC staining images and quantification of cells positive for MMP13 and NOS2 ( n = 5 mice per group) in Gls1 −/− mice and their Gls1 fl/fl control littermates at 8 weeks after sham and DMM surgery. ( E ) Safranin O/fast green staining and OARSI scores in joint sections of mice at 8 weeks after DMM surgery. Ad-Ctrl or Ad- Gls1 was intra-articularly injected once per week for 3 weeks beginning at 10 days after surgery ( n = 8 mice per group). Scale bars: 20 μm. The data are presented as box plots or as the mean ± SEM, and the dots represent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against SOX9 (Abcam, ab3697; 1:1,000); Collagen II (Abcam, ab85266; 1:1,000); MMP3 (Abcam, ab52915; 1:1,000); MMP13 (Proteintech, 18165-1-AP; 1:1,000); ADAMTS5 (Abcam, ab41037; 1:200); NOS2 (Abcam, ab178945; 1:1,000); SLC1A5 (Sigma, HPA035239; 1:1,000); GLS1 (Proteintech; 12855-1-ap; 1:1,000); GS (Abcam, ab176562; 1:1,000); NF-κB p65 (Santa Cruz Biotechnology; sc-8008; 1:1,000); lamin A/C (Santa Cruz Biotechnology; sc-6215; 1:5,000); phospho-IKKα/β (Cell Signaling Technology; 2694P; 1:1,000); IKKα/β (Abcam, ab178870; 1:1,000); phospho-IκBα (Affinity, AF2002; 1:1,000); IκBα (Affinity, AF5002; 1:1,000); TRAF6 (Santa Cruz Biotechnology; sc-8409; 1:200); K63 Ub (Cell Signaling Technology; 5621S; 1:1,000); and GAPDH (Abcam, ab8245; 1:2,000).

Techniques: Western Blot, Control, Staining, Immunohistochemistry, Injection

Journal: The Journal of Clinical Investigation

Article Title: α -Ketoglutarate protects against cartilage damage via epigenetically driven metabolic reprogramming in osteoarthritis models

doi: 10.1172/JCI172380

Figure Lengend Snippet: ( A ) IB analysis of K63-linked ubiquitination of TRAF6 in HEK293T cells transfected to express HA-TRAF6 with or without Flag-tagged K63-linked ubiquitin (Flag-K63-Ub) and MYC-tagged UBE2O (MYC-UBE2O). ( B ) Densitometry of the bands in A , showing the ubiquitination (Ub) of TRAF6, presented relative to results obtained with cells transfected to express empty vector, set as 100%. ( C ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 in IL-1β–stimulated chondrocytes in the presence of BPTES. Blots are representative of 3 independent experiments. ( D ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 that was immunoprecipitated and TRAF6 from IL-1β– and DM-αKG–stimulated chondrocytes. ( E ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 immunoprecipitated from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG. ( F ) Protein quantitative analysis of the nuclear p65 from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG for 60 minutes. ( G and H ) Quantitative analysis of qRT-PCR ( G ) and Western blot ( H ) analysis for the indicated anabolic and catabolic factors from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG. ( I ) IHC staining for pIκBα, p-p65, and quantification of pIκBα- and p-p65–positive cells at 25 weeks in mice fed a standard diet (SD) or HFD. ( J ) IHC staining for pIκBα, p-p65, and quantification of pIκBα- and p-p65–positive cells in Gls1 −/− mice and their Gls1 fl/fl control littermates after DMM surgery. veh, vehicle. ( K ) Schematic depicting the described findings: αKG inhibited the TRAF6 ubiquitination via inducing the expression of UBE2O in OA chondrocytes. The data are presented as box plots or as the mean ± SEM, and the dots represent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against SOX9 (Abcam, ab3697; 1:1,000); Collagen II (Abcam, ab85266; 1:1,000); MMP3 (Abcam, ab52915; 1:1,000); MMP13 (Proteintech, 18165-1-AP; 1:1,000); ADAMTS5 (Abcam, ab41037; 1:200); NOS2 (Abcam, ab178945; 1:1,000); SLC1A5 (Sigma, HPA035239; 1:1,000); GLS1 (Proteintech; 12855-1-ap; 1:1,000); GS (Abcam, ab176562; 1:1,000); NF-κB p65 (Santa Cruz Biotechnology; sc-8008; 1:1,000); lamin A/C (Santa Cruz Biotechnology; sc-6215; 1:5,000); phospho-IKKα/β (Cell Signaling Technology; 2694P; 1:1,000); IKKα/β (Abcam, ab178870; 1:1,000); phospho-IκBα (Affinity, AF2002; 1:1,000); IκBα (Affinity, AF5002; 1:1,000); TRAF6 (Santa Cruz Biotechnology; sc-8409; 1:200); K63 Ub (Cell Signaling Technology; 5621S; 1:1,000); and GAPDH (Abcam, ab8245; 1:2,000).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Immunohistochemistry, Control, Expressing

Journal: The Journal of Clinical Investigation

Article Title: α -Ketoglutarate protects against cartilage damage via epigenetically driven metabolic reprogramming in osteoarthritis models

doi: 10.1172/JCI172380

Figure Lengend Snippet: ( A ) Heatmap of amino acid content analyzed by LC-MS in sham- and DMM-operated knee cartilage ( n = 3 and 2, respectively) of mice at 8 weeks after surgery. ( B ) Heatmap of amino acid levels analyzed by LC-MS in chondrocytes treated with or without IL-1β (5 ng/mL) for 36 hours ( n = 4 per group). Ctrl, control. ( C ) Intracellular Gln levels in IL-1β–treated chondrocytes supplemented with Gln (4 or 8 mM) for 24 hours ( n = 3 per group). ( D and E ) Quantitative analysis of qRT-PCR ( n = 4 per group) ( D ) and Western blot ( E ) analysis for the indicated anabolic and catabolic factors in IL-1β–treated chondrocytes supplemented with Gln (4 or 8 mM) for 24 hours. ( F ) Schematic of Gln metabolism. ( G ) Western blot analysis for SLC1A5 and GLS1 in chondrocytes treated with or without IL-1β for 24 hours ( n = 3 per group). ( H ) Representative images of SLC1A5 and GLS1 immunofluorescence and mean intensity in joint cartilage of sham- or DMM-operated mice at 8 weeks after surgery ( n = 5 mice per group). Scale bars: 20 μm. ( I ) Representative images of SLC1A5 and GLS1 immunofluorescence and mean intensity in mice fed a standard diet (SD) and those fed an HFD ( n = 5 mice per group). Scale bars: 20 μm. ( J ) Representative images from safranin O staining and SLC1A5 and GLS1 immunofluorescence in undamaged ( n = 10), mildly damaged ( n = 10), and severely damaged (SevD) ( n = 7) human OA cartilage. Scale bars: 20 μm. ( K ) Gln and Glu levels in undamaged (UD) ( n = 10), mildly damaged (MD) ( n = 10) and SevD ( n = 7) human OA cartilage. The correlation between Gln levels and ICRS grading scores is shown in the right column. Scale bars: 200 μm. The data are presented as box plots or as the mean ± SEM, and the dots represent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Floxed Gls1 mice ( ) (catalog 017894) and Col2a1-CreERT2 mice ( ) (catalog 006774) were purchased from The Jackson Laboratory.

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: The Journal of Clinical Investigation

Article Title: α -Ketoglutarate protects against cartilage damage via epigenetically driven metabolic reprogramming in osteoarthritis models

doi: 10.1172/JCI172380

Figure Lengend Snippet: ( A and B ) mRNA ( A ) and Western blot ( B ) analysis of the indicated anabolic and catabolic factors in chondrocytes treated with BPTES or IL-1β alone or with combined BPTES and IL-1β for 24 hours. The culture medium contained 4 mM Gln. Blots are representative of 3 independent experiments. Ctrl, control. ( C ) Representative safranin O/fast green staining images and OARSI scores ( n = 8 mice per group) in Gls1 −/− mice and their Gls1 fl/fl control littermates at 8 weeks after sham and DMM surgery. ( D ) Representative MMP13 and NOS2 IHC staining images and quantification of cells positive for MMP13 and NOS2 ( n = 5 mice per group) in Gls1 −/− mice and their Gls1 fl/fl control littermates at 8 weeks after sham and DMM surgery. ( E ) Safranin O/fast green staining and OARSI scores in joint sections of mice at 8 weeks after DMM surgery. Ad-Ctrl or Ad- Gls1 was intra-articularly injected once per week for 3 weeks beginning at 10 days after surgery ( n = 8 mice per group). Scale bars: 20 μm. The data are presented as box plots or as the mean ± SEM, and the dots represent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Floxed Gls1 mice ( ) (catalog 017894) and Col2a1-CreERT2 mice ( ) (catalog 006774) were purchased from The Jackson Laboratory.

Techniques: Western Blot, Control, Staining, Immunohistochemistry, Injection

Journal: The Journal of Clinical Investigation

Article Title: α -Ketoglutarate protects against cartilage damage via epigenetically driven metabolic reprogramming in osteoarthritis models

doi: 10.1172/JCI172380

Figure Lengend Snippet: ( A ) IB analysis of K63-linked ubiquitination of TRAF6 in HEK293T cells transfected to express HA-TRAF6 with or without Flag-tagged K63-linked ubiquitin (Flag-K63-Ub) and MYC-tagged UBE2O (MYC-UBE2O). ( B ) Densitometry of the bands in A , showing the ubiquitination (Ub) of TRAF6, presented relative to results obtained with cells transfected to express empty vector, set as 100%. ( C ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 in IL-1β–stimulated chondrocytes in the presence of BPTES. Blots are representative of 3 independent experiments. ( D ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 that was immunoprecipitated and TRAF6 from IL-1β– and DM-αKG–stimulated chondrocytes. ( E ) Western blot analysis of K63-linked ubiquitination of endogenous TRAF6 immunoprecipitated from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG. ( F ) Protein quantitative analysis of the nuclear p65 from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG for 60 minutes. ( G and H ) Quantitative analysis of qRT-PCR ( G ) and Western blot ( H ) analysis for the indicated anabolic and catabolic factors from Ad-shCtrl and Ad-shUbe2o chondrocytes stimulated with IL-1β and DM-αKG. ( I ) IHC staining for pIκBα, p-p65, and quantification of pIκBα- and p-p65–positive cells at 25 weeks in mice fed a standard diet (SD) or HFD. ( J ) IHC staining for pIκBα, p-p65, and quantification of pIκBα- and p-p65–positive cells in Gls1 −/− mice and their Gls1 fl/fl control littermates after DMM surgery. veh, vehicle. ( K ) Schematic depicting the described findings: αKG inhibited the TRAF6 ubiquitination via inducing the expression of UBE2O in OA chondrocytes. The data are presented as box plots or as the mean ± SEM, and the dots represent biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Floxed Gls1 mice ( ) (catalog 017894) and Col2a1-CreERT2 mice ( ) (catalog 006774) were purchased from The Jackson Laboratory.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Immunohistochemistry, Control, Expressing