gls1 mice  (Jackson Laboratory)

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

Journal: Molecular Metabolism

Article Title: Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis

doi: 10.1016/j.molmet.2022.101655

Figure Lengend Snippet: Expression of Col3A1 and PLK1 in IPF fibroblasts transfected with non-targeting (NT) or GLS1 siRNA. IPF cells are transfected with NT or GLS1 siRNA in the presence of 2 mM glutamine. GLS1-silenced cells were cultured in presence of glutamine 2 mM with or without adding α-KG 2 mM. The cells were collected 48 h after transfection for western blots or real-time PCR. A/B: (A) Representative western-blotting to examine the protein levels for GLS1, Col3A1 and PLK1. (B) Densitometry analysis of Col3A1 and PLK1 associated signals: ration of Col3A1 and PLK1 to β-actin, as shown in A, n = 3 experimental replicates. ∗p < 0.05, GLS1 siRNA compared to NT siRNA, or GLS1 siRNA with 2 mM Gln plus α-KG. C/D: RNA expression of Col3A1 (C) or PLK1 (D) in two IPF fibroblast cell lines transfected with NT or GLS1 siRNA cultured in the presence of 2 mM glutamine with or without added α-KG as in A. Triangles or squares represent two different IPF patient-derived primary cells. Expressed values represent mean ± SD, 3 experimental replicates of each cell line. ∗p < 0.05, siRNA GLS1 vs NT, or vs siRNA GLS1 with 2 mM glutamine plus α-KG.

Article Snippet: GLS1 +/− heterozygous mice (#017956) were purchase from Jackson laboratory.

Techniques: Expressing, Transfection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Derivative Assay

Journal: Molecular Metabolism

Article Title: Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis

doi: 10.1016/j.molmet.2022.101655

Figure Lengend Snippet: Reduced fibrotic markers in the lungs of bleomycin-injured GLS1+/− heterozygous mice. A. Schematic timeline of animal studies. 8-week old WT or GLS1+/− heterozygous mice were subjected to saline or bleomycin injury. All mice were sacrificed 28 days after injury, samples were collected for assays. B. H&E stain of WT or GLS1+/− hets mice lungs subjected to saline or bleomycin. C. Semiquantitative Ashcroft score of lung sections with WT or GLS1+/− heterozygous mice subjected to saline or bleomycin on day 28 after injury. ∗p < 0.05, WT saline vs Bleo, or WT vs Hets of bleomycin injured mice (n = 3). D. Mice lung tissues were collected and the expression of GLS1, Col3A1, and PLK1 were examined by western blots for each of the following group: WT saline (n = 4), WT with bleomycin injury (n = 5), and Hets with bleomycin injury (n = 6). β-actin is the loading control. E-G. Densitometry of GLS1 (E), Col3A1 (F), and PLK1 (G) relative to β-actin (mean with standard error) as shown on the western blots in D. ∗p < 0.05, by t-test (WT saline vs bleo) or Kolmogorov–Smirnov test (WT bleo vs Hets bleo). H. Schematic diagram of possible mechanisms of the findings from this study. Glutamine control cell phenotype through an α-KG dependent and independent mechanisms to regulate gene expression.

Article Snippet: GLS1 +/− heterozygous mice (#017956) were purchase from Jackson laboratory.

Techniques: Saline, Staining, Expressing, Western Blot, Control, Gene Expression