Journal: Redox Biology
Article Title: The electrophilic metabolite of kynurenine, kynurenine-CKA, requires C151 in Keap1 to derepress Nrf2
doi: 10.1016/j.redox.2026.104009
Figure Lengend Snippet: Kynurenine-CKA is a Nrf2-dependent inducer of NQO1. (A) Human ARPE-19 cells were seeded into 96-well plates (10,000 cells/well) and treated 24 h post-seeding with either vehicle [0.1 % MeOH (v/v)] or increasing concentrations of: kynurenine, Kyn-CKA, the deaminated Kyn-CKA derivative Dean-Kyn-CKA, the reduced Kyn-CKA derivative Red-Kyn-CKA, or kynurenic acid (KynA). After 24 h, the specific enzyme activity of NQO1 was quantified in cell lysates. Data are shown as fold change in NQO1 specific enzyme activity relative to vehicle control. Values represent means of 8 biological replicates. Dose-response curves were fitted using a four-parameter logistic (4 PL) model in GraphPad Prism. (B) WT and Nrf2-KO BMDMs were seeded into six-well plates (1 × 10 6 /well), allowed to rest overnight, and then treated for 18 h with vehicle or Kyn-CKA (10 μM or 30 μM). The levels of mRNA for Nqo1 were determined by quantitative RT-PCR after normalization to the housekeeping control, B2m . Bars represent means ± SEM (n = 3 biologically independent replicates per condition), with individual data points shown. ∗, p < 0.05. (C,D) WT, Nrf2-KO and Keap1-KD BMDMs were seeded into six-well plates (1 × 10 6 /well), allowed to rest overnight, and subsequently treated with vehicle or Kyn-CKA (10 μM or 30 μM). Whole-cell lysates were collected after 3 h and 24 h and analysed by immunoblotting for Nrf2 at the 3 h timepoint (C) and for Nrf2, Keap1 and NQO1 at the 24h timepoint (D) , with GAPDH serving as a loading control.
Article Snippet: The TaqManTM Gene Expression Assay IDs (Thermo Fisher, Waltham, MA) were: Mm01253561 (for Nqo1 ); Mm00516005 (for Ho1); Mm01324400 (for Gclm ); Mm00434228 (for IL1b ); Mm00446190 (for IL6 ); Mm00441242 (for Mcp1 ); Mm00443252 (for Tnf ); Mm004405021 (for Nos2 ); Mm00437762 (for B2m ); Mm03024075 (for Hprt1 ); and 4352341E (for Actb ).
Techniques: Activity Assay, Control, Quantitative RT-PCR, Western Blot
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![Kynurenine-CKA is a Nrf2-dependent inducer of NQO1. (A) Human ARPE-19 cells were seeded into 96-well plates (10,000 cells/well) and treated 24 h post-seeding with either vehicle [0.1 % MeOH (v/v)] or increasing concentrations of: kynurenine, Kyn-CKA, the deaminated Kyn-CKA derivative Dean-Kyn-CKA, the reduced Kyn-CKA derivative Red-Kyn-CKA, or kynurenic acid (KynA). After 24 h, the specific enzyme activity of NQO1 was quantified in cell lysates. Data are shown as fold change in NQO1 specific enzyme activity relative to vehicle control. Values represent means of 8 biological replicates. Dose-response curves were fitted using a four-parameter logistic (4 PL) model in GraphPad Prism. (B) WT and Nrf2-KO BMDMs were seeded into six-well plates (1 × 10 6 /well), allowed to rest overnight, and then treated for 18 h with vehicle or Kyn-CKA (10 μM or 30 μM). The levels of mRNA for Nqo1 were determined by quantitative RT-PCR after normalization to the housekeeping control, <t>B2m</t> . Bars represent means ± SEM (n = 3 biologically independent replicates per condition), with individual data points shown. ∗, p < 0.05. (C,D) WT, Nrf2-KO and Keap1-KD BMDMs were seeded into six-well plates (1 × 10 6 /well), allowed to rest overnight, and subsequently treated with vehicle or Kyn-CKA (10 μM or 30 μM). Whole-cell lysates were collected after 3 h and 24 h and analysed by immunoblotting for Nrf2 at the 3 h timepoint (C) and for Nrf2, Keap1 and NQO1 at the 24h timepoint (D) , with GAPDH serving as a loading control.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8764/pmc12828764/pmc12828764__gr3.jpg)
