Journal: Genes & Diseases

Article Title: TIGAR promotes osteogenic differentiation and ameliorates glucocorticoid-induced osteoporosis via autophagy-Nrf2-ROS axis

doi: 10.1016/j.gendis.2025.101735

Figure Lengend Snippet: Effects of dexamethasone (Dex) on bone microstructure and TIGAR expression in femur of mice. The mouse model of osteoporosis was established by intramuscular injection of Dex (2 mg/kg and 5 mg/kg) for 8 weeks. (A) Representative three-dimensional reconstructive micro-CT images of trabecular bone and cortical bone from the control group and the Dex groups. (B) Bone mineral density (BMD), n = 5 mice. (C) Trabecular bone volume (BV/TV), n = 5 mice. (D) Trabecular number (Tb.N), n = 5 mice. (E) Trabecular separation (Tb.Sp), n = 5 mice. (F) Femur morphological analysis with H&E staining (scale bar, 200 μm). (G, H) Level of C-terminal cross linked peptide of type I collagen (CTX-I) ( n = 5 mice), (G) and N-terminal propeptide of type I collagen (PINP) (H) in mouse serum. (I, J) Confocal microscopy images showing the immunofluorescence staining of TIGAR expression in femur tissue and the quantification of the integrated optical density (IOD) per field. Scale bar, 100 μm; n = 3 mice. (K, L) Western blot analysis and quantification of TIGAR in femur tissue, n = 3. Data are shown as mean ± SEM. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. ∗∗∗ p < 0.001,∗∗ p < 0.01, ∗ p < 0.05. Dex-L, Dex dose of 2 mg/kg. Dex-H, Dex dose of 5 mg/kg.

Article Snippet: These are the primary antibodies that were used in this study: anti-mouse TIGAR (1:500, Santa Cruz, sc166290), anti-rabbit Runx2 (1:1000, Cell Signaling Technology, #12556), anti-rabbit sp7 (1:1000, Abcam, ab209484), anti-rabbit Nrf2 (1:1000, Zen-Bio, 380773), anti-rabbit keap1 (1:1000, Zen-Bio, R26935 ), anti-rabbit p62 (1:1000, Proteintech, 18420-1-AP), anti-rabbit LC3A/B (1:1000, Cell Signaling Technology, #12741), anti-rabbit HDAC1(1:1000, Cell Signaling Technology, #34589), and anti-mouse GAPDH (1:10000, ABclonal, AC002).

Techniques: Expressing, Injection, Micro-CT, Control, Staining, Confocal Microscopy, Immunofluorescence, Western Blot

Journal: Genes & Diseases

Article Title: TIGAR promotes osteogenic differentiation and ameliorates glucocorticoid-induced osteoporosis via autophagy-Nrf2-ROS axis

doi: 10.1016/j.gendis.2025.101735

Figure Lengend Snippet: Effects of dexamethasone (Dex) on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and TIGAR expression. (A) BMSCs were treated with Dex for 24, 48, and 72 h, and cells viability was analyzed with sulforhodamine B (SRB) cytotoxicity assay kit. (B–D) BMSCs were treated with different concentration of Dex for 48 h, and mRNA levels of runt-related transcription factor 2 (Runx2) , Sp7 transcription factor/osterix ( Sp7) , and alkaline phosphatase ( Alpl) were detected with qRT-PCR. (E–G) Western blotting analysis and quantification of Runx2 and Sp7 in BMSCs treated with different concentration of Dex for 48 h. (H) Alkaline phosphatase (ALP) staining and quantification of ALP-positive cells showed osteoblast activity in BMSCs treated with Dex on Day 9. (I) Alizarin Red S staining and quantification of mineralized nodules showed the role of Dex on osteogenic differentiation on Day 14. (J–L) BMSCs were treated with different concentration of Dex for 48 h; then the expression of TIGAR was detected by qRT-PCR (J) and western blotting (K and L). (M, N) Western blot analysis and quantification of TIGAR in osteogenic differentiation at different times (0, 3, 6, 9, and 12 d after addition of osteogenic differentiation medium). Data are shown as mean ± SEM. n = 3, biologically independent samples. One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was used to assess statistical significance. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: These are the primary antibodies that were used in this study: anti-mouse TIGAR (1:500, Santa Cruz, sc166290), anti-rabbit Runx2 (1:1000, Cell Signaling Technology, #12556), anti-rabbit sp7 (1:1000, Abcam, ab209484), anti-rabbit Nrf2 (1:1000, Zen-Bio, 380773), anti-rabbit keap1 (1:1000, Zen-Bio, R26935 ), anti-rabbit p62 (1:1000, Proteintech, 18420-1-AP), anti-rabbit LC3A/B (1:1000, Cell Signaling Technology, #12741), anti-rabbit HDAC1(1:1000, Cell Signaling Technology, #34589), and anti-mouse GAPDH (1:10000, ABclonal, AC002).

Techniques: Expressing, Cytotoxicity Assay, Concentration Assay, Quantitative RT-PCR, Western Blot, Staining, Activity Assay

Journal: Genes & Diseases

Article Title: TIGAR promotes osteogenic differentiation and ameliorates glucocorticoid-induced osteoporosis via autophagy-Nrf2-ROS axis

doi: 10.1016/j.gendis.2025.101735

Figure Lengend Snippet: Role of TIGAR on osteogenic differentiation under dexamethasone (Dex) treatment. (A–C) Bone marrow mesenchymal stem cells (BMSCs) transfected with TIGAR overexpression plasmid and with or without Dex treatment. TIGAR expression was detected by qRT-PCR (A) and western blotting (B), and (C) is the quantification of TIGAR protein expression. (D, E) mRNA expression of runt-related transcription factor 2 (Runx2) and Sp7 transcription factor/osterix ( Sp7) in different treatment groups. (F–H) Western blot analysis and quantification of Runx2 and Sp7 in different treatment groups. (I) Alkaline phosphatase (ALP) staining and quantification of ALP-positive cells showed osteoblast activity of BMSCs under different treatments. (J) Alizarin Red S staining and quantification of mineralized nodules showed the osteogenic differentiation ability of BMSCs under different treatments. (K–R) BMSCs transfected with TIGAR siRNA and with or without Dex treatment. Then, TIGAR, Runx2, and Sp7 mRNA and protein expression was detected by qRT-PCR and western blotting. (S) ALP staining and quantification of ALP-positive cells under different treatments. (T) Alizarin Red S staining and quantification of mineralized nodules under different treatments. Data are shown as mean ± SEM. n = 3, biologically independent samples. One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was used to assess statistical significance. ∗ p < 0.05, ∗∗ p < 0.01. NC, negative control. OE, TIGAR overexpression plasmid. Si, TIGAR siRNA.

Article Snippet: These are the primary antibodies that were used in this study: anti-mouse TIGAR (1:500, Santa Cruz, sc166290), anti-rabbit Runx2 (1:1000, Cell Signaling Technology, #12556), anti-rabbit sp7 (1:1000, Abcam, ab209484), anti-rabbit Nrf2 (1:1000, Zen-Bio, 380773), anti-rabbit keap1 (1:1000, Zen-Bio, R26935 ), anti-rabbit p62 (1:1000, Proteintech, 18420-1-AP), anti-rabbit LC3A/B (1:1000, Cell Signaling Technology, #12741), anti-rabbit HDAC1(1:1000, Cell Signaling Technology, #34589), and anti-mouse GAPDH (1:10000, ABclonal, AC002).

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Staining, Activity Assay, Negative Control

Journal: Genes & Diseases

Article Title: TIGAR promotes osteogenic differentiation and ameliorates glucocorticoid-induced osteoporosis via autophagy-Nrf2-ROS axis

doi: 10.1016/j.gendis.2025.101735

Figure Lengend Snippet: Protective role of TIGAR on dexamethasone (Dex)-induced bone loss. (A) Representative three-dimensional reconstructive micro-CT images of the trabecular and cortical bones from the control and Dex groups. (B) Bone mineral density (BMD). (C) Trabecular bone volume (BV/TV). (D) Trabecular number (Tb.N). (E) Trabecular separation (Tb.Sp). (F) Femur morphological analysis by H&E staining (scale bar, 200 μm). (G, H) Western blot analysis and quantification of TIGAR in femur tissue of wild type (WT) and TIGAR transgenic (TG). (I) Alkaline phosphatase (ALP) staining and quantification of ALP-positive cells showed osteoblast activity of bone marrow mesenchymal stem cells (BMSCs) derived from WT and TG mice. (J) Alizarin Red S staining and quantification of mineralized nodules showed the osteogenic differentiation ability of BMSCs derived from WT and TG mice. Data are shown as mean ± SEM. n = 3 mice. Statistical significance was assessed by two-way analysis of variance (ANOVA) with Tukey's multiple-comparisons test. ∗ p < 0.05, ∗∗ p < 0.01. WT, wild type; TG, TIGAR transgenic.

Article Snippet: These are the primary antibodies that were used in this study: anti-mouse TIGAR (1:500, Santa Cruz, sc166290), anti-rabbit Runx2 (1:1000, Cell Signaling Technology, #12556), anti-rabbit sp7 (1:1000, Abcam, ab209484), anti-rabbit Nrf2 (1:1000, Zen-Bio, 380773), anti-rabbit keap1 (1:1000, Zen-Bio, R26935 ), anti-rabbit p62 (1:1000, Proteintech, 18420-1-AP), anti-rabbit LC3A/B (1:1000, Cell Signaling Technology, #12741), anti-rabbit HDAC1(1:1000, Cell Signaling Technology, #34589), and anti-mouse GAPDH (1:10000, ABclonal, AC002).

Techniques: Micro-CT, Control, Staining, Western Blot, Transgenic Assay, Activity Assay, Derivative Assay

Journal: Genes & Diseases

Article Title: TIGAR promotes osteogenic differentiation and ameliorates glucocorticoid-induced osteoporosis via autophagy-Nrf2-ROS axis

doi: 10.1016/j.gendis.2025.101735

Figure Lengend Snippet: Mechanism of TIGAR preventing dexamethasone (Dex) from inhibiting osteoblastic differentiation. Bone marrow mesenchymal stem cells (BMSCs) were transfected with a TIGAR overexpression plasmid and treated with Dex. N-acetyl- l -cysteine (NAC, 10 mM), an antioxidant, was administrated in cells 1 h prior to Dex treatment. (A, B) ROS level was detected by dihydroethidium (DHE) and the quantification of the integrated optical density (IOD) per field. Scale bars, 200 μm. (C, D) BMSCs treated with the TIGAR overexpression plasmid and with or without Dex treatment were stained with Annexin-V and PI and detected using flow cytometry, and the apoptosis rate was quantified. (E, F) ROS level in femur of WT and TG mice with or without Dex treatment. Scale bars, 100 μm. (G, H) Cell apoptosis of femur tissue in vivo was detected by Tunel staining and the quantification of IOD per field. Scale bars, 100 μm. (I–K) mRNA expression of Sod1 , Gpx1 , and Cat of cells treated with Dex after TIGAR overexpression. (L) Relative NADPH/NADP + ratio. (M, N) Glutathione (GSH) and oxidative glutathione (GSSG) contents. In vitro : Data are shown as mean ± SEM. n = 3 biologically independent samples. One-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was used to assess statistical significance. ∗ p < 0.05, ∗∗ p < 0.01. NC, negative control. OE, TIGAR overexpression plasmid. In vivo : data were as the mean ± SEM, n = 3 mice. Statistical significance was assessed by two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. ∗ p < 0.05. WT, wild type; TG, TIGAR transgenic.

Article Snippet: These are the primary antibodies that were used in this study: anti-mouse TIGAR (1:500, Santa Cruz, sc166290), anti-rabbit Runx2 (1:1000, Cell Signaling Technology, #12556), anti-rabbit sp7 (1:1000, Abcam, ab209484), anti-rabbit Nrf2 (1:1000, Zen-Bio, 380773), anti-rabbit keap1 (1:1000, Zen-Bio, R26935 ), anti-rabbit p62 (1:1000, Proteintech, 18420-1-AP), anti-rabbit LC3A/B (1:1000, Cell Signaling Technology, #12741), anti-rabbit HDAC1(1:1000, Cell Signaling Technology, #34589), and anti-mouse GAPDH (1:10000, ABclonal, AC002).

Techniques: Transfection, Over Expression, Plasmid Preparation, Staining, Flow Cytometry, In Vivo, TUNEL Assay, Expressing, In Vitro, Negative Control, Transgenic Assay

Journal: Genes & Diseases

Article Title: TIGAR promotes osteogenic differentiation and ameliorates glucocorticoid-induced osteoporosis via autophagy-Nrf2-ROS axis

doi: 10.1016/j.gendis.2025.101735

Figure Lengend Snippet: TIGAR activated nuclear factor erythroid-2 related factor (Nrf2) to reduce dexamethasone (Dex)-induced oxidative stress through inducing autophagy. Bone marrow mesenchymal stem cells (BMSCs) were transfected with TIGAR overexpression plasmid and treated with Dex. (A, B) Immunofluorescence staining of Nrf2 of BMSCs and the quantification of the integrated optical density (IOD) per field. Scale bars, 50 μm. (C, D) Western blot analysis and quantification of Nrf2 expression in extracted nuclear proteins. (E, F) ROS level under Dex treatment in BMSCs with or without administration of 10 nM Nrf2 inhibitor (ML385) after transfecting with TIGAR overexpression plasmid, and the quantification of IOD per filed. Scale bars, 100 μm. BMSCs were treated with Dex and chloroquine (CQ) (20 μM) after transfecting with TIGAR overexpression plasmid. (G–I) Western blot analysis and quantification of p62 and LC3-II expression under different treatments. (J, K) Representative images of mCherry-GFP-LC3 puncta and number of autophagosomes (yellow) (analyzed by Pearson's correlation). Scale bars, 50 μm. (L–N) Western blot analysis and quantification of Nrf2 and kelch-associated protein 1 (Keap1) expression under different treatments. (O, P) The immunofluorescence staining of Nrf2 in BMSCs and the quantification of the IOD per field. Scale bars, 50 μm. (Q, R) Representative immunofluorescence images of LC3 and Keap1. Pearson's correlation of co-localization is shown in the bar graph format from the three independent experiments analyzed. Scale bars, 50 μm. (S, T) Representative images of ROS and the quantification of the IOD per field. Scale bars, 100 μm. Data are shown as mean ± SEM. n = 3, biologically independent samples. Two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test was used to assess statistical significance. ∗ p < 0.05, ∗∗ p < 0.01. NC, negative control. OE, TIGAR overexpression plasmid.

Article Snippet: These are the primary antibodies that were used in this study: anti-mouse TIGAR (1:500, Santa Cruz, sc166290), anti-rabbit Runx2 (1:1000, Cell Signaling Technology, #12556), anti-rabbit sp7 (1:1000, Abcam, ab209484), anti-rabbit Nrf2 (1:1000, Zen-Bio, 380773), anti-rabbit keap1 (1:1000, Zen-Bio, R26935 ), anti-rabbit p62 (1:1000, Proteintech, 18420-1-AP), anti-rabbit LC3A/B (1:1000, Cell Signaling Technology, #12741), anti-rabbit HDAC1(1:1000, Cell Signaling Technology, #34589), and anti-mouse GAPDH (1:10000, ABclonal, AC002).

Techniques: Transfection, Over Expression, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Expressing, Negative Control

Journal: JCI Insight

Article Title: TIGAR deficiency enhances cardiac resilience through epigenetic programming of Parkin expression

doi: 10.1172/jci.insight.200105

Figure Lengend Snippet: ( A and B ) qPCR analysis of TIGAR ( A ) and Parkin ( B ) mRNA in heart samples from WT and TKO mice, TKO mice injected with AAV9-cTnT-GFP control virus (TKO-GFP), AAV9-cTnT-TIGAR WT virus (TKO-TWT), or phosphatase-deficient TIGAR mutant virus (TKO-TMU), and PTKO mice. Data normalized to WT expression. ( C and D ) qPCR analysis of TIGAR ( C ) and Parkin ( D ) mRNA in heart samples from Myh6 Cre and hTKO mice, hTKO mice injected with AAV9-cTnT-GFP control virus (hTKO-GFP) or AAV9-cTnT-TIGAR WT virus (hTKO-TWT), and Parkin/TIGAR double-knockout (PTKO) mice. Data normalized to Myh6 Cre expression. ( E ) Western blot analysis of TIGAR (30 kDa) and Parkin (52 kDa) protein expression in heart samples from various genotypes as indicated (lanes 1–8), with vinculin (117 kDa) as loading control. Red arrows indicate TIGAR and Parkin bands. ( F and G ) qPCR analysis of TIGAR ( F ) and Parkin ( G ) mRNA in heart samples from WT mice treated with AAV9-GFP control or AAV9-cTnT-TIGAR shRNA. Data represent mean ± SD. Statistical significance was determined by 2-tailed Student’s t test. *** P < 0.005; n = 5 per group.

Article Snippet: Custom AAV9 vectors expressing WT Tigar ( NM_177003 ) and a phosphatase-deficient Tigar mutant were designed and manufactured by Charles River Laboratories for cardiac-specific gene delivery.

Techniques: Injection, Control, Virus, Mutagenesis, Expressing, Double Knockout, Western Blot, shRNA