Journal: Nature Communications

Article Title: Chondrocyte fatty acid oxidation drives osteoarthritis via SOX9 degradation and epigenetic regulation

doi: 10.1038/s41467-025-60037-4

Figure Lengend Snippet: a Quantification of 13 C-labeled acetyl-CoA levels in cartilage samples from mice that underwent DMM surgery, indicated in Fig. ( n = 4). b Western blot analysis of acetyl-lysine from whole cartilage lysate obtained from mice in Fig. . The immunoblotting results are representative of three independent experiments. c Bubble chart showing the top 20 enriched MFs among the upregulated acetylated proteins in HFD + DMM versus ND + DMM groups, as determined by a two-tailed Fisher’s exact test. d Heatmap showing acetylated lysine sites on proteins involved in lipid metabolic process in HFD + DMM versus ND + DMM groups. e Real-time relative-enzyme activity of Flag-tagged HADHA purified from C28/I2 cells treated with FFA or TSA + NAM. The area under the curve (AUC) was generated by Prism 9.5.1. Relative HADHA activity was calculated at 20 min ( n = 4). f Real-time relative-enzyme activity of Flag-tagged wild-type HADHA or HADHA K728R purified from HADHA -KO C28/I2 cells treated with FFA or BSA. The AUC was generated using Prism 9.5.1. Relative HADHA activities were calculated at 30 min ( n = 4). g Mitochondrial fuel oxidation analysis. HADHA-KO C28/I2 cells overexpressing wild-type HADHA or HADHA K728R were both pretreated with FFA for 24 h before loading. The cells were then sequentially treated with UK5099, BPTES, and Eto ( n = 5 for HADHA WT group; n = 6 for HADHA K728R group). h Molecular interactions of wild-type, K728R, and K728ac HADHA with the substrate. The SASA and binding energy were calculated. a , e right, f right, and g right Data are minimum to maximum: each point represents an individual biological replicate, box from 25th to 75th percentiles; center line at median; tukey whiskers, as determined via unpaired two-tailed t -test ( a and g right) or one-way ANOVA followed by Dunnett’s multiple-comparisons test ( e right), or two-way ANOVA followed by Tukey’s multiple-comparisons test ( f right). e left, f left, and g left Data are mean ± SD, as determined via one-way ANOVA followed by Dunnett’s multiple-comparisons test. Source data are provided as a Source Data file.

Article Snippet: Hadha fl/fl , Hadhb fl/fl , and Hadha CKI/CKI (p. K728R Conditional Knock in) mice were purchased from Cyagen Biology Technology.

Techniques: Labeling, Western Blot, Two Tailed Test, Activity Assay, Purification, Generated, Binding Assay

Journal: Nature Communications

Article Title: Chondrocyte fatty acid oxidation drives osteoarthritis via SOX9 degradation and epigenetic regulation

doi: 10.1038/s41467-025-60037-4

Figure Lengend Snippet: a Immunoblot detection of ACAN, COL2A1, SOX9, MMP3, MMP13, and HADHA in mouse chondrocytes transfected with Hadha siRNAs. All cells were treated with 200 μM FFA. b mRNA expression levels of Hadha in mouse chondrocytes infected with a lentivirus encoding Hadha short hairpin RNA #2 ( n = 6). c , d Micromass culture ( c ) or 3D-agarose culture ( d ) and alcian blue staining of murine chondrocytes infected with a lentivirus encoding Hadha short hairpin RNA #2 ( n = 6). All cells were treated with 200 μM FFA. e 3D-agarose culture and alcian blue staining of murine chondrocytes treated with FFA or FFA + TMZ ( n = 6). f Immunoblot detection of COL2A1, SOX9, MMP13, and HADHA in HADHA -KO C28/I2 cells overexpressing HADHA or harboring the empty vector. All cells were treated with 200 μM FFA. g Immunoblot detection of ACAN, COL2A1, SOX9, MMP13, and HADHA in HADHA -KO C28/I2 cells overexpressing wild-type HADHA, HADHA K728R mutant, or harboring the empty vector. All cells were treated with 200 μM FFA. h Safranin O/Fast green staining and IHC staining for HADHA in knee joints of Hadha fl/fl and Hadha fl/fl ; Acan-creER T2 mice that underwent DMM or sham surgery. Samples were collected 12 weeks after surgery ( n = 8). i Safranin O/Fast green staining; IHC staining for HADHA and HADHA K728ac; and IF staining for COL2A1 and MMP13 in knee joints from Hadha K728R CKI/CKI and Hadha K728R CKI/CKI ; Acan-creER T2 mice that underwent DMM surgery and fed HFD or ND. Samples were collected 8 weeks after surgery ( n = 8). b , h , and i Data are mean ± SD, as determined via unpaired two-tailed t -test, or nonparametric Mann–Whitney test. c – e Data are minimum to maximum: each point represents an individual biological replicate, box from 25th to 75th percentiles; center line at median; tukey whiskers, as determined via unpaired two-tailed t -test ( c , d ) or one-way ANOVA followed by Tukey’s multiple-comparisons test ( e ). The immunoblotting data are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Hadha fl/fl , Hadhb fl/fl , and Hadha CKI/CKI (p. K728R Conditional Knock in) mice were purchased from Cyagen Biology Technology.

Techniques: Western Blot, Transfection, Expressing, Infection, shRNA, Staining, Plasmid Preparation, Mutagenesis, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Chondrocyte fatty acid oxidation drives osteoarthritis via SOX9 degradation and epigenetic regulation

doi: 10.1038/s41467-025-60037-4

Figure Lengend Snippet: a Sox9 mRNA levels in mouse chondrocytes treated with FFA ( n = 6). b , c Immunoblot analysis of SOX9 in primary murine chondrocytes treated as indicated. d ATP production after FFA exposure in HADHA- KO C28/I2 cells overexpressing wild-type HADHA or HADHA K728R or harboring the empty vector ( n = 3). e Immunoblot of p-ACC/ACC, p-AMPK/AMPK, SOX9, and HADHA in Hadha fl/fl murine chondrocytes infected with Cre or adenovirus vector 72 h before harvesting. f Immunoblot detection of ubiquitinated Flag-tagged SOX9 overexpressed in C28/I2 cells via Flag immunoprecipitation. g Heatmap showing the results of UPLC-MS/MS analysis for identifying phosphorylation sites on the SOX9 protein purified from C28/I2 cells. The presence, absence, or lack of detection of modifications at the specified sites in the SOX9 protein are denoted by ‘1’, ‘−1’, and ‘0’ respectively. h Immunoblot detection of AMPK phosphorylates consensus motif (p-AMPK motif) in Flag-tagged wild-type, and SOX9 ST-A mutant overexpressed in C28/I2 cells using an anti-p-AMPK-motif antibody via Flag immunoprecipitation. i Immunoblot detection of ubiquitinated, Flag-tagged wild-type, ST-A, and ST-D SOX9 overexpressed in C28/I2 cells via Flag immunoprecipitation. ST-A and ST-D denote SOX9 variants with six serine/threonine residues mutated to alanine and aspartate residues, respectively. j Co-immunoprecipitation of SOX9 with TRIM9 in HEK293T cells treated with FFA or BSA. k Co-immunoprecipitation of Flag-tagged wild-type SOX9 or SOX9 mutants with HA-TRIM9 in HEK293T cells. l Molecular docking results demonstrating the interaction between wild-type or ST-phosphorylated SOX9 and TRIM9. m Immunoblot detection of TRIM9 and SOX9 in HEK293T cells transfected with TRIM9 siRNAs. n Immunoblot detection of p-ACC/ACC, p-AMPK/AMPK, SOX9, and HADHA in HADHA -KO C28/I2 cells overexpressing wild-type HADHA, HADHA K728R mutant, or harboring the empty vector. o IF staining for p-ACC and IHC staining for SOX9 in mouse-knee cartilage tissues indicated in Fig. ( n = 8). Data are mean ± SD, as determined by one-way ANOVA followed by Dunnett’s multiple-comparisons test ( a , d ), or unpaired two-tailed t -test ( o ). The immunoblotting data are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: Hadha fl/fl , Hadhb fl/fl , and Hadha CKI/CKI (p. K728R Conditional Knock in) mice were purchased from Cyagen Biology Technology.

Techniques: Western Blot, Plasmid Preparation, Infection, Immunoprecipitation, Tandem Mass Spectroscopy, Phospho-proteomics, Purification, Mutagenesis, Transfection, Staining, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: Chondrocyte fatty acid oxidation drives osteoarthritis via SOX9 degradation and epigenetic regulation

doi: 10.1038/s41467-025-60037-4

Figure Lengend Snippet: Fatty acids that accumulate in chondrocytes enter the FAO pathway and lead to acetyl-CoA accumulation, which reshapes the chondrocyte protein acetylation profile. Mitochondrial acetyl-CoA facilitates the acetylation of HADHA, enhancing its enzymatic activity and creating a positive feedback loop that further amplifies FAO. Concurrently, excessive FAO suppresses AMPK activity, resulting in reduced phosphorylation and increased ubiquitination-mediated degradation of SOX9. In parallel, nuclear acetyl-CoA alters histone acetylation patterns, promoting transcriptional activation of ECM catabolic genes such as MMP13 and ADAMTS7 while suppressing ECM anabolic genes including ACAN and COL2A1. These combined effects disrupt ECM turnover and accelerate cartilage degradation. Pharmacological inhibition of FAO using TMZ effectively interrupts this pathological cascade and mitigates OA progression (created by figdraw.com).

Article Snippet: Hadha fl/fl , Hadhb fl/fl , and Hadha CKI/CKI (p. K728R Conditional Knock in) mice were purchased from Cyagen Biology Technology.

Techniques: Activity Assay, Phospho-proteomics, Ubiquitin Proteomics, Activation Assay, Inhibition

Journal: eLife

Article Title: Salmonella- induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis

doi: 10.7554/eLife.93125

Figure Lengend Snippet: ( A ) Metabolic gene expression data of S . Typhimurium-infected RAW 264.7 macrophages at 2 hr, 6 hr, and 16 hr time points of infection through nanoString. Data is representative of N = 2, n = 1. ( B ) Ppard qPCR expression data in S . Typhimurium-infected RAW 264.7 macrophages at the indicated time points of infection. Data is representative of N = 3, n = 2. ( C ) Lactate estimation assay of S . Typhimurium-infected RAW 264.7 macrophages at the initial time point of 2 hr and at the late time point of 16 hr post-infection. Data is representative of N = 3, n = 3. Unpaired two-tailed Student’s t -test was performed to obtain the p-values (**p<0.01, *p<0.05). ( D, E ) Lactate estimation assay of S . Typhimurium-infected RAW 264.7 macrophages upon Sirt1 ( D ) or Sirt3 ( E ) knockdown condition at 16 hr post-infection. Data is representative of N = 3, n = 3. Unpaired two-tailed Student’s t -test was performed to obtain the p-values. ( F ) Immunoblotting of host glycolytic (PGK), TCA cycle (PDHA1), and fatty acid oxidation (HADHA, ACOX-1) proteins under Sirt1 and Sirt3 knockdown condition of S . Typhimurium-infected RAW 264.7 macrophages at 16 hr post-infection. ( G ) Fatty acid oxidation (FAO) assay of uninfected (UI) and infected (STM) RAW 264.7 macrophages under Sirt1 or Sirt3 knockdown or inhibitor treatment. N = 2, n = 2 (** p<0.01, *p<0.05). ( H ) FAO assay of uninfected (UI) and infected RAW 264.7 macrophages under infection with wildtype S . Typhimurium (STM WT), SPI-1 (∆ invC ), or SPI-2 (∆ ssaV and ∆steE ) m utants of S . Typhimurium. Data is representative of N = 2,n = 2 (**p<0.01, * p<0.05). Figure 5—source data 1. Original files for western blot analysis displayed in . Figure 5—source data 2. File containing original uncropped western blots for , indicating relevant bands and treatments.

Article Snippet: Antibody , HADHA monoclonal , ABclonal , Cat# A5346 , 1:1000 dilution.

Techniques: Gene Expression, Infection, Expressing, Two Tailed Test, Knockdown, Western Blot

Journal: eLife

Article Title: Salmonella- induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis

doi: 10.7554/eLife.93125

Figure Lengend Snippet: ( A ) qRT PCR-mediated gene expression analysis of several host metabolic genes in Sirt1 or Sirt3 knockdown RAW 264.7 macrophages at 6 hr post-infection. Data is representative of N = 3,n = 3. Unpaired two-tailed Student’s t -test was performed to obtain the p-values (*p<0.05). ( B ) qRT PCR-mediated gene expression analysis of several host metabolic genes in SIRT1 or SIRT3 inhibitor-treated (1 µM) peritoneal macrophages isolated from 6- to 8-week-old C57BL/6 male mice liver harvested on fifth day post-thioglycolate injection. Data is representative of N = 3,n = 3. Unpaired two-tailed Student’s t -test was performed to obtain the p-values (****p<0.0001, *** p<0.001, **p<0.01, *p<0.05). ( C ) Lactate estimation assay of S . Typhimurium-infected peritoneal macrophages upon SIRT1 or SIRT3 catalytic inhibition at 2 hr, 6 hr, and 16 hr post-infection. Data is representative of N = 3,n = 3. Unpaired two-tailed Student’s t -test was performed to obtain the p-values (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05). ( D ) Immunoblotting of host glycolytic (PGK, PFK) and fatty acid oxidation (HADHA, ACOX-1) proteins under SIRT1 (EX-527) and SIRT3 (3TYP) catalytic inhibitor-treated condition in S. Typhimurium-infected peritoneal macrophages (isolated from thioglycolate-treated adult male C57BL/6 mice) at 16 hr post-infection. Data is representative of N = 2, n = 1. ( E ) qRT PCR- mediated gene expression profiling of several host metabolic genes in SIRT1 or SIRT3 inhibitor-treated 6- to 8-week-old C57BL/6 male mice liver harvested on fifth day post-infection of S. Typhimurium (10 7 CFU units/animal). Unpaired two-tailed Student’s t -test was performed to obtain the p-values. Data is representative of N = 3, n = 3 (****p<0.0001, ***p<0.001, ** p<0.01, *p<0.05). ( F ) qRT PCR-mediated gene expression profiling of several host metabolic genes in SIRT1 or SIRT3 inhibitor-treated 6- to 8-week-old C57BL/6 male mice spleen harvested on fifth day post-infection of S. Typhimurium (10 7 CFU units/animal). Unpaired two-tailed Student’s t -test was performed to obtain the p-values. Data is representative of N = 3, n = 3 (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05). Figure 5—figure supplement 1—source data 1. Original files for western blot analysis displayed in . Figure 5—figure supplement 1—source data 2. File containing original uncropped western blots for , indicating relevant bands and treatments.

Article Snippet: Antibody , HADHA monoclonal , ABclonal , Cat# A5346 , 1:1000 dilution.

Techniques: Quantitative RT-PCR, Gene Expression, Knockdown, Infection, Two Tailed Test, Isolation, Injection, Inhibition, Western Blot

Journal: eLife

Article Title: Salmonella- induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis

doi: 10.7554/eLife.93125

Figure Lengend Snippet: List of primers.

Article Snippet: Antibody , HADHA monoclonal , ABclonal , Cat# A5346 , 1:1000 dilution.

Techniques: Sequencing

Journal: eLife

Article Title: Salmonella- induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis

doi: 10.7554/eLife.93125

Figure Lengend Snippet:

Article Snippet: Antibody , HADHA monoclonal , ABclonal , Cat# A5346 , 1:1000 dilution.

Techniques: Recombinant, shRNA, Western Blot, Immunoprecipitation, Lactate Assay, Protease Inhibitor