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: Journal of Cellular and Molecular Medicine

Article Title: Spontaneous colitis in IL‐10‐deficient mice was ameliorated via inhibiting glutaminase1

doi: 10.1111/jcmm.14471

Figure Lengend Snippet: Gls 1 is highly expressed in the intestines of CD patients and Il‐10 –/– mice. Immunohistochemical staining with an antibody that recognizes Gls1 was performed on the intestines of control and CD patients (A). The quantitative analysis presented in (B) shows Gls1 expression in the intestines of CD (n = 13) and control patients (n = 17). (C,D) Western blot analysis of Gls1 in the intestinal mucosa in the intestines of CD patients (inflamed and uninflamed areas) and control patients. (E,F) The expression of Gls1 in the intestines of Il‐10 –/– mice and WT mice (n = 8 in each group). CD, Crohn's disease; Gls1, glutaminase 1; IOD, integrated optical density; WT, wild‐type. The data are presented as the relative IOD ± SD. *** P < 0.001

Article Snippet: A total of 60 μg/mice BPTES (Gls1 inhibitor; Selleck Chemicals, USA) or dimethyl sulfoxide (DMSO) in PBS was intraperitoneally administered twice a week for 4 weeks as reported previously.

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Spontaneous colitis in IL‐10‐deficient mice was ameliorated via inhibiting glutaminase1

doi: 10.1111/jcmm.14471

Figure Lengend Snippet: Systemic delivery of BPTES ameliorates experimental colitis in Il‐10 –/– mice. The 15‐wk‐old Il‐10 –/– mice were intraperitoneally treated with BPTES (60 μg/mice) or DMSO in PBS for 4 wk. A, The disease activity index (DAI) in BPTES‐treated Il‐10 –/– mice and untreated Il‐10 –/– mice was scored weekly. B, Bodyweight changes in the BPTES‐treated Il‐10 –/– mice and untreated Il‐10 –/– mice. C, Intestinal tissue sections were stained with haematoxylin and eosin for histological analysis under light microscopy (scale bar: 50 μm). The histological inflammation score (D) was analysed as described in the Section . E, ELISAs of IL‐17A, TNF‐α and IFN‐γ protein expression in intestinal tissue homogenate. F, Concentration of Gln in intestinal homogenate. (G,H) The relative mRNA and protein levels of Gls1 in intestinal mucosa. The data are expressed as the mean ± SD (n = 8 for each group). * P < 0.05, ** P < 0.01, *** P < 0.001. BPTES, bis‐2‐ (5‐phenylacetamido‐1,2, 4‐thiadiazol‐2‐yl) ethyl sulphide; Gls1, glutaminase 1; IFN, interferon; IL, interleukin; TNF, tumour necrosis factor

Article Snippet: A total of 60 μg/mice BPTES (Gls1 inhibitor; Selleck Chemicals, USA) or dimethyl sulfoxide (DMSO) in PBS was intraperitoneally administered twice a week for 4 weeks as reported previously.

Techniques: Activity Assay, Staining, Light Microscopy, Expressing, Concentration Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Spontaneous colitis in IL‐10‐deficient mice was ameliorated via inhibiting glutaminase1

doi: 10.1111/jcmm.14471

Figure Lengend Snippet: Gls1 regulate different T‐cell subset responses in IL‐10 −/− mice involve inactivating mTORC1. Total protein extracts were prepared from intestinal tissue, and the protein levels of p‐p70 S6K, p70 S6K, p‐4E‐BP1 and 4E‐BP1 were analysed by western blot; β‐actin was used as an internal reference A,. Densitometric assays of all protein band intensities (B,C). The data are expressed as the mean ± SD (n = 8 per group). Gls1, glutaminase 1; NS, no significance; * P < 0.05, ** P < 0.01

Article Snippet: A total of 60 μg/mice BPTES (Gls1 inhibitor; Selleck Chemicals, USA) or dimethyl sulfoxide (DMSO) in PBS was intraperitoneally administered twice a week for 4 weeks as reported previously.

Techniques: Western Blot

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Role of Glutaminase 2 in Promoting CD4+ T Cell Production of Interleukin-2 by Supporting Antioxidant Defense in Systemic Lupus Erythematosus

doi: 10.1002/art.42112

Figure Lengend Snippet: Epigenetic influence of glutaminase 2 (GLS2) on reactive oxygen species (ROS) levels and interleukin-2 (IL-2) production in murine CD4+ T cells. A-F, Naive CD4+ T cells from wild-type B6 mice were cultured with anti-CD3/anti-CD28 and transfected with empty vector (Empty), GLS1-overexpression plasmid (GLS1 OE), or GLS2-overexpression plasmid (GLS2 OE) on day 1. On day 3, CellRox Green Reagent was used to detect ROS (n = 8 samples per group) (A), Liperfluo was used to detect lipid peroxidation (n = 6 per group) (B), staining with MitoTracker Deep Red and Green was performed to assess mitochondrial polarization and mass, respectively (n = 4 per group) (C), a dual luciferase assay was used to assess IL-2 promoter activity (n = 5 per group) (D), an enzyme-linked immunosorbent assay was used to measure the IL-2 concentration in culture supernatants (n = 7 per group) (E), and an EZ DNA Methylation Kit was used to assess CpG DNA methylation (n = 7 per group) (F). In all transfection experiments, DsRed+CD4+ T cells were analyzed. Bars show the mean ± SEM; symbols represent individual samples. * = P < 0.05; ** = P < 0.01, by one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. MFI = mean fluorescence intensity; NS = not significant. G, Alignment of the murine and human genes encoding IL-2 shows conserved noncoding sequences (CNS) (red) that were determined as regions of interest for further analysis of CpG DNA methylation. CNS1–CNS3 are located in the proximal promoter, which spans 2 kb, and CNS4 is located in the 3′-untranslated region (3′-UTR).

Article Snippet: Site-directed mutagenesis for substituting the PDZ domain–binding motif in the mouse GLS2-overexpression vector with the corresponding sequence in the mouse Gls1 gene was performed using the Q5 site-directed mutagenesis kit (New England Biolabs) with the following primers: 5′-TTGCTATAAGGATCCGCCCCTCTCCCT-3′ and 5′-CCCGTCGAGATTCTCTTTGGACAGGGTCTCAGC-3′.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Over Expression, Staining, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, DNA Methylation Assay, Fluorescence

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Role of Glutaminase 2 in Promoting CD4+ T Cell Production of Interleukin-2 by Supporting Antioxidant Defense in Systemic Lupus Erythematosus

doi: 10.1002/art.42112

Figure Lengend Snippet: Role of the PDZ domain–binding motif in GLS2-mediated regulation of IL-2 and ROS levels in mice. A, Structures of GLS1 (isoform KGA), GLS2 (isoform GAB), and mutated GLS2, in which the PDZ domain–binding motif at the C-terminus (red) was substituted with that of GLS1, are shown. B–E, Naive CD4+ T cells from wild-type B6 mice were cultured with anti-CD3/anti-CD28 and transfected with empty vector, GLS2 OE, or mutated GLS2–overexpression plasmid (GLS2 with mutation OE) on day 1. On day 3, assessment of ROS (n = 6 samples per group) (B), lipid peroxidation (n = 6 per group) (C), mitochondrial polarization (n = 4 per group) (D), and Il2 promoter activity (n = 5 per group) (E) was performed. In all transfection experiments, DsRed+ CD4+ T cells were analyzed. Bars show the mean ± SEM; symbols represent individual samples. * = P < 0.05; ** = P < 0.01, by one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. See Figure 1 for other definitions.

Article Snippet: Site-directed mutagenesis for substituting the PDZ domain–binding motif in the mouse GLS2-overexpression vector with the corresponding sequence in the mouse Gls1 gene was performed using the Q5 site-directed mutagenesis kit (New England Biolabs) with the following primers: 5′-TTGCTATAAGGATCCGCCCCTCTCCCT-3′ and 5′-CCCGTCGAGATTCTCTTTGGACAGGGTCTCAGC-3′.

Techniques: Binding Assay, Cell Culture, Transfection, Plasmid Preparation, Over Expression, Mutagenesis, Activity Assay

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Role of Glutaminase 2 in Promoting CD4+ T Cell Production of Interleukin-2 by Supporting Antioxidant Defense in Systemic Lupus Erythematosus

doi: 10.1002/art.42112

Figure Lengend Snippet: Effect of GLS2 on IL-2 production and ROS levels in CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus (SLE). A and B, GLS2 and actin expression in purified CD4+T cells from 16-week-old MRL/MpJ-Faslpr/J (MRL/lpr) and MRL.MpJ (control) mice (n = 6 samples per group) (A) and healthy control participants and patients with SLE (n = 14 per group) (B) were analyzed by Western blotting. C–F, Naive CD4+ T cells from 16-week-old MRL.lpr mice, MRL.MpJ mice, healthy control participants, and patients with SLE were cultured with anti-CD3/anti-CD28. Cells from MRL.MpJ mice and healthy controls were transfected with empty vector, and cells from MRL.lpr mice (C and D) and patients with SLE (E and F) were transfected with empty vector, GLS1 OE, or GLS2 OE. On day 3, assessment of ROS (n = 6 per group) and Il2 and IL2 promoter activity (n = 6 per group) was performed. In all transfection experiments, DsRed+CD4+ T cells were analyzed. Bars show the mean ± SEM; symbols represent individual samples. * = P < 0.05; ** = P < 0.01, by Student’s paired t-test and one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. See Figure 1 for other definitions.

Article Snippet: Site-directed mutagenesis for substituting the PDZ domain–binding motif in the mouse GLS2-overexpression vector with the corresponding sequence in the mouse Gls1 gene was performed using the Q5 site-directed mutagenesis kit (New England Biolabs) with the following primers: 5′-TTGCTATAAGGATCCGCCCCTCTCCCT-3′ and 5′-CCCGTCGAGATTCTCTTTGGACAGGGTCTCAGC-3′.

Techniques: Expressing, Purification, Control, Western Blot, Cell Culture, Transfection, Plasmid Preparation, Activity Assay