Journal: Redox Biology

Article Title: IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD + de novo synthesis and curbing endothelial senescence

doi: 10.1016/j.redox.2025.103695

Figure Lengend Snippet: The expression of the endothelial NAD + de novo synthesis enzyme IDO1 is reduced after lower limb ischemia in aged mice. (A) Scheme depicting the experiment for RNA-seq and NAD + -target metabolomics of young and aged mice following HLI. (B) Diagram showing the pathway of NAD + synthesis and consuming. (C) Volcano plot of transcriptional alterations in hindlimb muscle ECs 7 days after HLI. (D) RNA-seq analysis of NAD + metabolism related enzymes in hindlimb muscle ECs 7 days after HLI. Young group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (E) Heatmap of the NAD + related metabolites in hindlimb muscle ECs 7 days after HLI. Young group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (F) Western blot analysis and quantification of IDO1 in hindlimb muscle ECs 7 days after HLI. Young group was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗∗∗ P < 0.001. (G) Violin plots of log-transformed gene expression of Ido1 in cell populations of hindlimb muscle from young and aged mice following HLI. (H) Violin plots of log-transformed gene expression of IDO1 in cell populations of lower-limb muscle of PAD patients.

Article Snippet: Mouse IL-17A protein (Catalog #HY- P70753 ), mouse IL-17F protein (Catalog #HY- P72584 ), mouse IL-17A/F protein (Catalog #HY- P77707 ), mouse IDO1 protein (Catalog #HY- P72616 ), IgG (Catalog #HY-P99002), brodalumab (Catalog #HY-P9925, an anti-interleukin-17-receptor IgG2 monoclonal antibody), and 666–15 (Catalog #HY-101120, CREB phosphorylation inhibitor) were purchased from Med-ChemExpress.

Techniques: Expressing, RNA Sequencing, Comparison, Western Blot, Transformation Assay, Gene Expression

Journal: Redox Biology

Article Title: IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD + de novo synthesis and curbing endothelial senescence

doi: 10.1016/j.redox.2025.103695

Figure Lengend Snippet: Ido1 deficiency leads to ECs senescence and impairs neovascularization in the ischemic hindlimb muscle of young mice. (A) Design showing the experiment for ECs senescence and vasculogenic phenotypes in Ido1 −/− mice. (B) Mass spectrometry analysis of NAD + related metabolites in hindlimb muscle ECs 7 days after HLI. WT group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001, ns: not significant. (C) Western blot analysis and quantification of P16 in hindlimb muscle ECs 7 days after HLI. WT group was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (D–F) RT-qPCR quantification of p16 (D), p21 (E) and Lmnb1 (F) in hindlimb muscle ECs 7 days after HLI. WT group was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (G, H) ROS levels (G) and NO levels (H) quantification in hindlimb muscle ECs 7 days after HLI. Data of WT group in G was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗∗∗ P < 0.0001. (I) Immunofluorescence staining for CD31, α-SMA and KI67 in ischemic adductor muscles 7 days after HLI is shown. Scale bar, 200 μm. (J) Quantification of the percent of KI67 + arterial ECs in total arterial ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (K) Immunofluorescence staining for CD31 and KI67 in ischemic gastrocnemius muscles 7 days after HLI is shown. Scale bar, 200 μm. (L) Quantification of the percent of KI67 + CD31 + cells in total capillary ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (M) Immunofluorescence staining for CD31, α-SMA and γ-H2AX in ischemic adductor muscles 7 days after HLI is shown. Scale bar, 100 μm. (N) Quantification of the percent of γ-H2AX + arterial ECs in total arterial ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (O) Immunofluorescence staining for CD31 and γ-H2AX in ischemic gastrocnemius muscles 7 days after HLI is shown. Scale bar, 100 μm. (P) Quantification of the percent of γ-H2AX + CD31 + cells in total capillary ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (Q and R) Representative laser Doppler images (Q) of hindlimbs of mice and quantification (R) of relative perfusion recovery as the blood flow ratio of the left limb to that of the right limb in WT and Ido1 −/− mice before and after HLI at the indicated time points. The areas under the curves of relative perfusion are shown, Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001. (S) Representative micro-CT images of ischemic hindlimbs 21 days after HLI. Scale bar, 3 mm. (T) Quantification of micro-CT analysis of arterial vasculature in ischemic hindlimbs. Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (U) Representative whole-mounted images of ischemic gracilis 21 days after HLI. (V) Quantification of the diameter of pre-existed collateral arteries (6 points per artery). Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗ P < 0.001. (W) Immunofluorescence staining for CD31 in ischemic gastrocnemius muscles 21 days after HLI is shown. Scale bar, 200 μm. (X) Quantification of the CD31-positive area in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001.

Article Snippet: Mouse IL-17A protein (Catalog #HY- P70753 ), mouse IL-17F protein (Catalog #HY- P72584 ), mouse IL-17A/F protein (Catalog #HY- P77707 ), mouse IDO1 protein (Catalog #HY- P72616 ), IgG (Catalog #HY-P99002), brodalumab (Catalog #HY-P9925, an anti-interleukin-17-receptor IgG2 monoclonal antibody), and 666–15 (Catalog #HY-101120, CREB phosphorylation inhibitor) were purchased from Med-ChemExpress.

Techniques: Mass Spectrometry, Comparison, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Muscles, Micro-CT

Journal: Redox Biology

Article Title: IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD + de novo synthesis and curbing endothelial senescence

doi: 10.1016/j.redox.2025.103695

Figure Lengend Snippet: IL-17A/F represses ECs Ido1 transactivation and NAD + biosynthesis, and facilitates ECs senescence in the ischemic hindlimb muscle of young mice. A) Schematic diagram for testing methods was indicated. (B) Bar plot of the most prominent categories by KEGG pathway (left) and GO analysis (right). (C) Western blot analysis and quantification of IDO1 in hindlimb muscle ECs 7 days after HLI. PBS group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (D) RT-qPCR quantification of Ido1 in hindlimb muscle ECs 7 days after HLI. PBS group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05. (E) Western blot analysis and quantification of IDO1 and P16 in hindlimb muscle ECs 7 days after HLI. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001, ns: not significant. (F–I) RT-qPCR quantification of Ido1 (F), p16 (G) , p21 (H) and Lmnb1 (I) in hindlimb muscle ECs 7 days after HLI. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (J–L) ROS (J), NAD + (K) and NO (L) levels quantification in in hindlimb muscle ECs 7 days after HLI. Data of Con group in J was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (M) Venn diagram of IDO1 transcriptional factors between mouse and human. (N) Western blot analysis and quantification of P-CREB and total-CREB in hindlimb muscle ECs 7 days after HLI. Young group was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗∗∗ P < 0.001. (O, P) Western blot analysis and quantification of P-CREB, total-CREB (T-CREB) (O), IDO1(P) and P16 (P) in hindlimb muscle ECs 7 days after HLI. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (Q–T) RT-qPCR quantification of Ido1 (Q), p16 (R) , p21 (S) and Lmnb1 (T) in hindlimb muscle ECs 7 days after HLI. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns: not significant. (U–W) ROS (U), NAD + (V) and NO (W) levels quantification in in hindlimb muscle ECs 7 days after HLI. Data of Con group in U was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant.

Article Snippet: Mouse IL-17A protein (Catalog #HY- P70753 ), mouse IL-17F protein (Catalog #HY- P72584 ), mouse IL-17A/F protein (Catalog #HY- P77707 ), mouse IDO1 protein (Catalog #HY- P72616 ), IgG (Catalog #HY-P99002), brodalumab (Catalog #HY-P9925, an anti-interleukin-17-receptor IgG2 monoclonal antibody), and 666–15 (Catalog #HY-101120, CREB phosphorylation inhibitor) were purchased from Med-ChemExpress.

Techniques: Western Blot, Comparison, Quantitative RT-PCR

Journal: Redox Biology

Article Title: IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD + de novo synthesis and curbing endothelial senescence

doi: 10.1016/j.redox.2025.103695

Figure Lengend Snippet: IL-17A/F counteracts Endothelial Ido1 overexpression-induced neovascularization under conditions of lower limb ischemia. (A) Scheme showing the experiment for rescuing the IL-17A/F-treated young mice by endothelial Ido1 overexpression. (B and C) Representative laser Doppler images (B) of hindlimbs of mice and quantification (C) of relative perfusion recovery as the blood flow ratio of the left limb to that of the right limb in AAV-NC + PBS, AAV-NC + IL-17A/F, AAV- Ido1 + PBS and AAV- Ido1 + IL-17A/F mice before and after HLI at the indicated time points. The areas under the curves of relative perfusion are shown, Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗AAV-NC + IL-17A/F vs. AAV-NC + PBS; # AAV- Ido1 + IL-17A/F vs. AAV-NC+ IL-17A/F, #/ ∗ P < 0.05, ### P < 0.001, ∗∗∗∗/ #### P < 0.0001. (D) Representative micro-CT images of ischemic hindlimbs 21 days after HLI. Scale bar, 3 mm. (E) Quantification of micro-CT analysis of arterial vasculature in ischemic hindlimbs. Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (F) Representative whole-mounted images of ischemic gracilis 21 days after HLI. (G) Quantification of the diameter of pre-existed collateral arteries (6 points per artery). One-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (H) Immunofluorescence staining for CD31 in ischemic gastrocnemius muscles 21 days after HLI is shown. Scale bar, 200 μm. (I) Quantification of the CD31-positive area in muscle. One-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001.

Article Snippet: Mouse IL-17A protein (Catalog #HY- P70753 ), mouse IL-17F protein (Catalog #HY- P72584 ), mouse IL-17A/F protein (Catalog #HY- P77707 ), mouse IDO1 protein (Catalog #HY- P72616 ), IgG (Catalog #HY-P99002), brodalumab (Catalog #HY-P9925, an anti-interleukin-17-receptor IgG2 monoclonal antibody), and 666–15 (Catalog #HY-101120, CREB phosphorylation inhibitor) were purchased from Med-ChemExpress.

Techniques: Over Expression, Comparison, Micro-CT, Immunofluorescence, Staining, Muscles

Journal: Redox Biology

Article Title: IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD + de novo synthesis and curbing endothelial senescence

doi: 10.1016/j.redox.2025.103695

Figure Lengend Snippet: IDO1 protein alleviates ECs senescence, and boosts ECs migration, tube formation and proliferation in vitro . (A) Schematic diagram for HUVECs treated with H 2 O 2 for 24 h and incubated with or without IDO1 for 24 h. (B and C) Cell senescence was determined using SA-β-Gal staining, and the SA-β-Gal positive cells were quantified. Scale bar, 75 μm. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001, ns: not significant. (D) P16 levels were measured using western blot analysis, and the mean gray value was quantified. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001, ns: not significant. (E–G) P16 (E) , P21 (F) and LMNB1 (G) levels were analyzed using RT-qPCR analysis. Con group was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.001, ns: not significant. (H–J) ROS (H), NAD + (I) and NO (J) levels were quantified. Data of Con group in H was set as 1. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (K and L) Cell migration was evaluated using a transwell assay, and the migrated cells were quantified. Scale bar, 75 μm. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001, ns: not significant. (M and N) Endothelial tube formation in Matrigel was assessed, and the total branch length was quantified. Scale bar, 75 μm. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05, ns: not significant. (O and P) Cell proliferation was analyzed using EdU staining, and the EdU positive cells were quantified. Scale bar, 75 μm. One-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001, ns: not significant.

Article Snippet: Mouse IL-17A protein (Catalog #HY- P70753 ), mouse IL-17F protein (Catalog #HY- P72584 ), mouse IL-17A/F protein (Catalog #HY- P77707 ), mouse IDO1 protein (Catalog #HY- P72616 ), IgG (Catalog #HY-P99002), brodalumab (Catalog #HY-P9925, an anti-interleukin-17-receptor IgG2 monoclonal antibody), and 666–15 (Catalog #HY-101120, CREB phosphorylation inhibitor) were purchased from Med-ChemExpress.

Techniques: Migration, In Vitro, Incubation, Staining, Comparison, Western Blot, Quantitative RT-PCR, Transwell Assay

Journal: Redox Biology

Article Title: IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD + de novo synthesis and curbing endothelial senescence

doi: 10.1016/j.redox.2025.103695

Figure Lengend Snippet: IDO1 protein enhances the neovascularization in aged mice following lower limb ischemia. (A) Scheme illustrating the experiment of aged mice administrated with IDO1 protein. (B) Mass spectrometry analysis of NAD + related metabolites in hindlimb muscle ECs 7 days after HLI. Aged + PBS group was set as 1. Two-way ANOVA with Tukey multiple comparison test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (C) Western blot analysis and quantification of P16 in hindlimb muscle ECs 7 days after HLI. Aged + PBS group was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗∗∗ P < 0.001. (D–F) RT-qPCR quantification of p16 (D), p21 (E), Lmnb1 (F) in hindlimb muscle ECs 7 days after HLI. Aged + PBS group was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01. (G and H) ROS (G) and NO (H) levels quantification in hindlimb muscle ECs 7 days after HLI. Data of Aged + PBS group in G was set as 1. Unpaired two-tail t -test, n = 3 per group, data are mean ± SEM, ∗∗ P < 0.001, ∗∗∗ P < 0.001. (I) Immunofluorescence staining for CD31, α-SMA and KI67 in ischemic adductor muscles 7 days after HLI is shown. Scale bar, 200 μm. (J) Quantification of the percent of KI67 + arterial ECs in total arterial ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (K) Immunofluorescence staining for CD31 and KI67 in ischemic gastrocnemius muscles 7 days after HLI is shown. Scale bar, 200 μm. (L) Quantification of the percent of KI67 + CD31 + cells in total capillary ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (M) Immunofluorescence staining for CD31, α-SMA and γ-H2AX in ischemic adductor muscles 7 days after HLI is shown. Scale bar, 100 μm. (N) Quantification of the percent of γ-H2AX + arterial ECs in arterial ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (O) Immunofluorescence staining for CD31 and γ-H2AX in ischemic gastrocnemius muscles 7 days after HLI is shown. Scale bar, 100 μm. (P) Quantification of the percent of γ-H2AX + CD31 + cells in total capillary ECs in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. (Q and R) Representative laser Doppler images of hindlimbs of mice and quantification of relative perfusion recovery as the blood flow ratio of the left limb to that of the right limb in Aged + PBS and Aged + IDO1 mice before and after HLI at the indicated time points. The areas under the curves of relative perfusion are shown, Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001. (S) Representative micro-CT images of ischemic hindlimbs 21 days after HLI. Scale bar, 3 mm. (T) Quantification of micro-CT analysis of arterial vasculature in ischemic hindlimbs. Two-way ANOVA with Tukey multiple comparison test, n = 6 per group, data are mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, ns: not significant. (U) Representative whole-mounted images of ischemic gracilis 21 days after HLI. (V) Quantification of the diameter of pre-existed collateral arteries (6 points per artery). Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗ P < 0.01. (W) Immunofluorescence staining for CD31 in ischemic gastrocnemius muscles 21 days after HLI is shown. Scale bar, 200 μm. (X) Quantification of the CD31-positive area in muscle. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001.

Article Snippet: Mouse IL-17A protein (Catalog #HY- P70753 ), mouse IL-17F protein (Catalog #HY- P72584 ), mouse IL-17A/F protein (Catalog #HY- P77707 ), mouse IDO1 protein (Catalog #HY- P72616 ), IgG (Catalog #HY-P99002), brodalumab (Catalog #HY-P9925, an anti-interleukin-17-receptor IgG2 monoclonal antibody), and 666–15 (Catalog #HY-101120, CREB phosphorylation inhibitor) were purchased from Med-ChemExpress.

Techniques: Mass Spectrometry, Comparison, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Muscles, Micro-CT

Journal: Redox Biology

Article Title: IDO1 improves postischemic neovascularization in aged mice by boosting endothelial NAD + de novo synthesis and curbing endothelial senescence

doi: 10.1016/j.redox.2025.103695

Figure Lengend Snippet: Plasma IDO1 levels are lower in elderly PAD patients and correlate with PAD severity, onset risk, and cardiovascular outcome. ( A ) Schematic diagram for the process of sample collection and correlation analysis between plasma IDO1 levels and clinical indicators. ( B ) ELISA analysis of IDO1 levels in plasma from young and aged mic at 7 days following HLI. Unpaired two-tail t -test, n = 6 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. ( C ) ELISA analysis of IDO1 levels in plasma from young <65 years: and elderly ≥65 years: patients with PAD. Unpaired two-tail t -test, n = 30 per group, data are mean ± SEM, ∗∗∗∗ P < 0.0001. ( D – K ) Spearman correlation between plasma concentration of IDO1 and ABI ( D ), HDL ( E ), PCSK9 ( F ), NLR ( G ), hs-CRP ( H ), Fibrinogen ( I ), D-dimer ( J ) and NT-proBNP ( K ). n = 30 per group. ( L ) Schematic diagram for the changes in plasma IDO1 levels and clinical indicators between young and elderly PAD patients.

Article Snippet: Mouse IL-17A protein (Catalog #HY- P70753 ), mouse IL-17F protein (Catalog #HY- P72584 ), mouse IL-17A/F protein (Catalog #HY- P77707 ), mouse IDO1 protein (Catalog #HY- P72616 ), IgG (Catalog #HY-P99002), brodalumab (Catalog #HY-P9925, an anti-interleukin-17-receptor IgG2 monoclonal antibody), and 666–15 (Catalog #HY-101120, CREB phosphorylation inhibitor) were purchased from Med-ChemExpress.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancer cell

Article Title: Tumor-Repopulating Cells Induce PD-1 Expression in CD8 + T Cells by Transferring Kynurenine and AhR Activation.

doi: 10.1016/j.ccell.2018.02.005

Figure Lengend Snippet: Figure 3. IFN-g Upregulates the Expression of IDO1 and SLC1A5 to Boost Kyn Production (A) The mRNA expression of IDO1 in bulk cells (B16, A375, or HepG2) or TRCs treated with PBS or IFN-g (10 ng/mL) for 24 hr. (B) OVA-B16 TRCs were co-cultured without or with anti-CD3/CD28 bead-activated OT-1 cells for 24 hr. The expression of Ido1 was determined by qPCR. (C and D) Ido1 shRNA-(Sh1 or Sh2)-OVA B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OT-1 T cells. After 4 hr, TRC apoptosis (C) or after 24 hr, percentage of PD-1+CD8+ cells (D) was analyzed by flow cytometry. Iso, isotype control. (E) OVA-B16, A375, or HepG2 bulk cells or TRCs were treated with PBS or IFN-g (10 ng/mL) for 24 hr. The expression of SLC1A5, SLC7A5, or SLC7A8 was determined by qPCR. (F) Tryptophan (Trp) levels were determined by ELISA in cell lysates of Slc1a5 shRNA (Sh1 or Sh2)-B16 TRCs treated with or without IFN-g (10 ng/mL) for 24 hr. (G and H) Slc1a5 shRNA (Sh1 or Sh2)-OVA B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OT-1 cells. After 4 hr, TRC apoptosis (G) or after 24 hr, percentage of PD-1+CD8+ cells (H) was analyzed by flow cytometry. Iso, isotype control. (I) B16 bulk cells were transfected with vector or Flag-SLC1A5 plasmid. SLC1A5 expression and tryptophan consumption were determined by western blot (left) and ELISA (right). (J) B16 bulk cells expressing vector or SLC1A5 were treated with PBS or IFN-g (10 ng/mL) for 24 hr. Kyn levels in supernatants were determined by ELISA. *p < 0.05, **p < 0.01, by one-way ANOVA (A, C–H, and J) or Student’s t test (B and I). The data represent means ± SEM of three independent experiments. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Ido1 Mouse shRNA Origene TL511273 Slc1a5 Mouse shRNA Origene TL511273 Software and Algorithms FlowJo (version7.6.1) FlowJo, LLC https://www.flowjo.com/ solutions/flowjo StepOne software (version 2.2) ThermoFisher Scientific https://www.thermofisher.com/ order/catalog/product/4376600 ImageJ (version 1.44p) NIH Image https://imagej.nih.gov/ij/ Genomatix PromoterInspector Genomatix http://www.genomatix.de/ GraphPad Prism (version 6.0) GraphPad Software Inc https://www.graphpad.com/ scientificsoftware/prism/

Techniques: Expressing, Cell Culture, shRNA, Cytometry, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Western Blot

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Indoleamine 2,3-dioxygenase 1 and programmed cell death-ligand 1 co-expression correlates with aggressive features in lung adenocarcinoma.

doi: 10.1016/j.ejca.2018.06.020

Figure Lengend Snippet: Fig. 1. Representative images of immunohistochemical staining of IDO1 in human placental tissue and surgically resected specimens from patients with primary lung adenocarcinoma. (A) Human placental tissue showing strong cytoplasmic and membrane staining in endo- thelial cells. Scale bar: 100 mm. (B) Lung adenocarcinoma showing positive cytoplasmic and membrane staining. Scale bar: 100 mm. (CeE) Typical IDO1 staining of lung adenocarcinoma at TPS of <1% (C), 1e50% (D), and 50% (E). Scale bars: 500 mm. IDO1, indoleamine 2,3-dioxygenase 1; TPS, tumour proportion score.

Article Snippet: The sections were then incubated with a 1:200 dilution of mouse antihuman IDO1 monoclonal antibody (clone UMAB126; Origene Technologies, Rockville, MD) at 4 C overnight.

Techniques: Immunohistochemical staining, Staining, Membrane

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Indoleamine 2,3-dioxygenase 1 and programmed cell death-ligand 1 co-expression correlates with aggressive features in lung adenocarcinoma.

doi: 10.1016/j.ejca.2018.06.020

Figure Lengend Snippet: Fig. 2. KaplaneMeier curves showing survival of patients with primary lung adenocarcinoma according to IDO1 expression. (A) Disease- free survival and (B) overall survival according to IDO1 expression status determined by the 1% cut-off value. (C) Disease-free survival and (D) overall survival according to IDO1 expression status, as determined by the 50% cut-off value. IDO1, indoleamine 2,3-dioxygenase 1; TPS, tumour proportion score.

Article Snippet: The sections were then incubated with a 1:200 dilution of mouse antihuman IDO1 monoclonal antibody (clone UMAB126; Origene Technologies, Rockville, MD) at 4 C overnight.

Techniques: Expressing

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Indoleamine 2,3-dioxygenase 1 and programmed cell death-ligand 1 co-expression correlates with aggressive features in lung adenocarcinoma.

doi: 10.1016/j.ejca.2018.06.020

Figure Lengend Snippet: Fig. 3. KaplaneMeier curves showing survival of patients with primary lung adenocarcinoma according to IDO1 and PD-L1 expression. We adopted cut-off values of 1% for IDO1 and PD-L1 expression in this analysis. (A) Disease-free survival and (B) overall survival of patients expressing the indicated combinations of IDO1 and PD-L1. IDO1, indoleamine 2,3-dioxygenase 1; PD-L1, programmed cell death-ligand 1.

Article Snippet: The sections were then incubated with a 1:200 dilution of mouse antihuman IDO1 monoclonal antibody (clone UMAB126; Origene Technologies, Rockville, MD) at 4 C overnight.

Techniques: Expressing