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