Journal: The Journal of Biological Chemistry

Article Title: Tryptophan metabolites 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) increase oxidative stress and impair osteoblastic bone formation

doi: 10.1016/j.jbc.2026.111174

Figure Lengend Snippet: Tryptophan and its downstream metabolites activate AhR transcriptional activity . A , luciferase assay results showing activation of AhR transcriptional activity by tryptophan and its downstream metabolites. AhR transcriptional activity after inhibiting IDO1 with PF-06840003 (10 μM) ( B ) and Kyn-3-monooxygenase with JM6 (5 μM) ( C ) and treating ST2 cells with tryptophan and KYN respectively. D , Time course qPCR experiment showing Cyp1a1 expression in C2C12 cells following KYN treatment. C2C12 cells were treated with tryptophan metabolites for 3 h ( E ) and 24 h ( F ) and the mRNA levels of AhR target genes were measured via RT-qPCR. Multiple group comparison was performed by one way ANOVA and Fisher’s least significant difference (LSD) post hoc testing and bars with different superscript letters are significantly ( p < 0.05) different from one another (n ≥ 3 biological replicates for each condition). Cyp1a1, cytochrome p450 1a1; Cyp1b1, cytochrome p450 1b1; Ahrr, Aryl hydrocarbon repressor. # indicates significant difference versus vehicle control at p < 0.05 (#), p < 0.01 (##), p < 0.001 (###) as shown by one sample t-tests versus vehicle controls. AhR, aryl hydrocarbon receptor; Kyn, kynurenine.

Article Snippet: Immortalized murine ST2 (obtained from Leibniz Institute DSMZ, #ACC333) and C2C12 (obtained from ATCC, #CRL-1772) cell lines were used for in vitro studies.

Techniques: Activity Assay, Luciferase, Activation Assay, Expressing, Quantitative RT-PCR, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: Tryptophan metabolites 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) increase oxidative stress and impair osteoblastic bone formation

doi: 10.1016/j.jbc.2026.111174

Figure Lengend Snippet: 3-hydroxy-kynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) induce DNA damage leading to senescence at low concentrations and apoptotic cell death at higher concentration . DNA damage was evaluated after treating ST2 cells with 3HK ( A ), 3HAA ( B ), Tryptophan (Tryp) ( C ) and Kyn ( D ) then staining the cells with Gamma H2AX antibody. ( E ) ST2 cells were treated with tryptophan metabolites (100 μM) or doxorubicin as a positive control (1 μM) for 24 h, fixed and stained for β-galactosidase activity to reflect senescence, β-galactosidase positive cells were quantified using the manual cell counting function of ImageJ. MTT assays were used to measure cell viability after treating ( F ) C2C12 and ( G ) ST2 cells with tryptophan metabolites for 24 h. H , representative images showing that 3HK (400 μM) and 3HAA (300 μM) induced apoptosis in C2C12 cells as indicated by positive TUNEL staining ( left ); ImageJ was utilized to quantify TUNEL labeling from n ≥ 3 biological replicates ( right ), and the results were analyzed by Student’s t test. The scale bar represents 50 μm. MTT assay showing that scavenging ROS with N-Acetylcysteine rescued ( I ) 3HK and ( J ) 3HAA-induced cell death. Multiple group comparison was performed by one way ANOVA and Fisher’s LSD post hoc testing and bars with different superscript letters are significantly ( p < 0.05) different from one another (n ≥ 3 biological replicates for each condition). 3HAA, 3-hydroxyanthranilic acid; 3HK, 3-hydroxy-kynurenine.

Article Snippet: Immortalized murine ST2 (obtained from Leibniz Institute DSMZ, #ACC333) and C2C12 (obtained from ATCC, #CRL-1772) cell lines were used for in vitro studies.

Techniques: Concentration Assay, Staining, Positive Control, Activity Assay, Cell Counting, TUNEL Assay, Labeling, MTT Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Tryptophan metabolites 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) increase oxidative stress and impair osteoblastic bone formation

doi: 10.1016/j.jbc.2026.111174

Figure Lengend Snippet: HK and 3HAA induce oxidative stress in mesenchymal lineage cells . Amplex red assay measuring whole cell ROS production after 6 h of treatment with ( A and C ) 3HK and ( B and D ) 3HAA in the presence and absence of the ROS scavenger N-acetyl cysteine (5 mM). One sample t test was used to compare treatment conditions with their vehicle control # indicates significant difference versus veh. at p < 0.05 (#), p < 0.01 (##), p < 0.001 (###). Amplex red assay was used to measure mitochondrial ROS ( E ) while tetramethylrhodamine methyl ester measured transmembrane potential ( F ) after 6 h treatment of ST2 cells with tryptophan metabolites. Multiple group comparison was done by one way ANOVA and Fisher’s LSD post hoc testing and bars with different superscript letters are significantly ( p < 0.05) different from one another (n ≥ 3 biological replicates for each condition). The expression of the antioxidant enzyme Hmox1 and ( G and H ) its transcription factor regulator Nrf-2 ( I and J ) was increased following a 24 h treatment of ST2 cells with 3HK and 3HAA. One sample t test was used to compare treatment conditions with their vehicle control; # indicates significant difference versus veh. at p < 0.05 (#), p < 0.01 (##), p < 0.001 (###). 3HAA, 3-hydroxyanthranilic acid; 3HK, 3-hydroxy-kynurenine; ROS, reactive oxygen species.

Article Snippet: Immortalized murine ST2 (obtained from Leibniz Institute DSMZ, #ACC333) and C2C12 (obtained from ATCC, #CRL-1772) cell lines were used for in vitro studies.

Techniques: Amplex Red Assay, Control, Comparison, Expressing