ido1  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Adverse pro-tumorigenic effects of IDO1 catalytic inhibitors mediated by the non-enzymatic function of IDO1 in tumor cells

doi: 10.3389/fimmu.2025.1680896

Figure Lengend Snippet: IDO1 catalytic inhibitors increase IDO1 protein expression in human tumor cell lines. (A) Kynurenine released by SKOV-3, FTC-133, B-CPAP, and RC-K8 cells treated with EPA, LIN, and NAV (1 µM) for 24 h Vehicle-treated cells were used as a control (ctrl). Results are shown as kynurenine fold change (FC) of treated versus respective ctrl cells (dotted line, 1-fold). (B) IDO1 protein expression in SKOV-3, FTC-133, B-CPAP, and RC-K8 cells treated as in (A) . β-tubulin expression was used as a normalizer. One representative immunoblot of five is shown. The IDO1/β-tubulin ratio of scanning densitometry analysis is reported as FC of treated versus respective ctrl cells (dotted line, 1-fold). (C) Real-time PCR analysis of IDO1 transcript in SKOV-3, FTC-133, B-CPAP, and RC-K8 cells treated as in (A) , normalized to the expression of ACTB and reported as relative to the results in the respective ctrl cells (dotted line, 1-fold). Data in (A–C) are mean ± SD of independent experiments ( A , B : N = 5; C : N = 3) and were analyzed by one-way ANOVA followed by post hoc Bonferroni’s test. Statistical significance shown in (A) is representative of all comparisons (EPA, LIN, or NAV versus ctrl).

Article Snippet: IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4ETM, Cell Signaling Technology, Danvers, MA, USA), whereas the SHP-2 protein level was detected using a mouse monoclonal anti -SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Adverse pro-tumorigenic effects of IDO1 catalytic inhibitors mediated by the non-enzymatic function of IDO1 in tumor cells

doi: 10.3389/fimmu.2025.1680896

Figure Lengend Snippet: IDO1 catalytic inhibitors prolong the IDO1 protein half-life in FTC-133 cells. (A, B) Cycloheximide chase assay followed by immunoblot analysis of IDO1 protein expression in FTC-133 cells pretreated with cycloheximide (50 μg/mL) for 1 h (as time 0) and then exposed to EPA, LIN, and NAV (1 µM) for a short (up to 8 h) (A) or long (up to 16 h) (B) kinetics. For each time point, vehicle-treated cells were used as a control (ctrl), whereas β-tubulin expression was used as a normalizer. One immunoblot representative of three is shown. The exponential decay regression analysis of the IDO1/β-tubulin protein ratio is expressed as a percentage (%) of time 0 (time 0 = 100%; dotted line, 50%). The half-life ( t 1/2 , in hours) and the degradation speed (K, in hours −1 ) of IDO1 protein are reported as mean ± SD and shown in the table ( B , lower right panel). (C) Kynurenine released by FTC-133 cells treated as in (A, B) , measured at the last time points of the kinetics. Results are shown as kynurenine FC of treated versus respective ctrl cells (dotted line, 1-fold). Data in (A–C) are mean ± SD of independent experiments ( A , B : N = 3; C : N = 5) and were analyzed by two-way ANOVA followed by post-hoc Bonferroni’s test. Statistical significance shown in (C) is representative of all comparisons (EPA, LIN, and NAV versus the respective ctrl).

Article Snippet: IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4ETM, Cell Signaling Technology, Danvers, MA, USA), whereas the SHP-2 protein level was detected using a mouse monoclonal anti -SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Adverse pro-tumorigenic effects of IDO1 catalytic inhibitors mediated by the non-enzymatic function of IDO1 in tumor cells

doi: 10.3389/fimmu.2025.1680896

Figure Lengend Snippet: IDO1 catalytic inhibitors enhance the non-enzymatic function of IDO1 in FTC-133 cells. (A) p-Tyr immunoprecipitation (IP) and IDO1 immunoblot analysis in FTC-133 cells treated with EPA, LIN, and NAV (1 µM) for 4 h Vehicle-treated cells were used as control (ctrl), whereas WCLs were used as controls of input protein expression, and a negative control (n.c.; i.e., only resin) was included. (B) Phosphorylated Src (p-Src) and total Src immunoblot analysis of FTC-133 cells treated as in (A) . In (A, B) β-tubulin or actin were used as normalizers and quantitative data from densitometric analysis (pTyr-IDO1 or pSrc/Src ratios, respectively) are reported as FC of treated versus ctrl cells (1-fold), above the corresponding bands. (C) Tyr-phosphorylation of rhIDO1 incubated with rhSrc in the presence of ATP (100 µM) for 60 min. rhIDO1 was preconditioned with EPA, NAV, and LIN (10 μM) for 30 min prior to rhSrc exposure, while vehicle-preconditioned rhIDO1 was used as a ctrl. The p-Tyr/IDO1 ratio and the p-Src/Src ratio were calculated by densitometric analysis and reported as FC of EPA/LIN/NAV-conditioned samples versus the ctrl sample (60 min; dotted line, 1-fold). (D) Kinase activity of rhSrc incubated as in (C) and reported as FC of relative light unit (RLU) measured in EPA/LIN/NAV-conditioned samples versus the ctrl sample (dotted line, 1-fold). (E) In situ PLA between IDO1 and Src performed in FTC-133 cells treated with EPA, LIN, and NAV (1 μM) for 1 h (F) In situ PLA between IDO1 and SHP-2 performed in FTC-133 cells treated with EPA, LIN, and NAV (1 μM) for 16 h In (E, F) vehicle-treated cells were used as ctrl. For each condition, a representative field of view is shown (60× magnification; scale bar: 10 µm) and the number of red spots, indicating the single IDO1/Src (E) or IDO1/SHP-2 (F) interaction, is reported as a function of cell number in each field of view. For (A–C) one representative immunoblot of three is shown. Data in (A–F) are mean ± SD of independent experiments ( N = 3) and were analyzed by one-way ANOVA followed by post-hoc Bonferroni’s test.

Article Snippet: IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4ETM, Cell Signaling Technology, Danvers, MA, USA), whereas the SHP-2 protein level was detected using a mouse monoclonal anti -SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Immunoprecipitation, Western Blot, Control, Expressing, Negative Control, Phospho-proteomics, Incubation, Activity Assay, In Situ

Journal: Frontiers in Immunology

Article Title: Adverse pro-tumorigenic effects of IDO1 catalytic inhibitors mediated by the non-enzymatic function of IDO1 in tumor cells

doi: 10.3389/fimmu.2025.1680896

Figure Lengend Snippet: Pro-tumorigenic effects of IDO1 catalytic inhibitors in FTC-133 cells. (A) Analysis of the wound closure (black dotted lines) on FTC-133 cells in the presence of EPA, LIN, and NAV (1 µM) or vehicle alone (ctrl) over time. For each reported time point, one representative image of three is shown (10× magnification; scale bar: 100 µm), and data are reported as wound closure percentage (%) of respective time 0 (time 0 = 0%; dotted line, 50%). (B) Transwell migration assay performed on FTC-133 cells exposed to EPA, LIN, and NAV (1 µM) in serum-free medium. Vehicle-treated cells were used as ctrl, while vehicle-treated cells cultured over serum-free medium were included as a negative ctrl (n.c.). For each condition, a representative field of view is shown (10× magnification; scale bar: 400 µm), and cell migration is reported as percentage (%) of cell-covered area, calculated in each field of view. (C) Soft agar colony formation assay in FTC-133 cells exposed to EPA, LIN, and NAV (1 µM) or vehicle alone (ctrl) once a week. For each condition, the colony size (µm) of 100 representative colonies is reported, whereas one representative image of three is shown (10× magnification; scale bar: 100 µm). Data in (A–C) are mean ± SD of independent experiments ( A , C : N = 3; B : N = 5). Data in (A ) and (B, C) were analyzed by two-way or one-way ANOVA followed by post-hoc Bonferroni’s test, respectively.

Article Snippet: IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4ETM, Cell Signaling Technology, Danvers, MA, USA), whereas the SHP-2 protein level was detected using a mouse monoclonal anti -SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Transwell Migration Assay, Cell Culture, Migration, Soft Agar Assay

Journal: Frontiers in Immunology

Article Title: Adverse pro-tumorigenic effects of IDO1 catalytic inhibitors mediated by the non-enzymatic function of IDO1 in tumor cells

doi: 10.3389/fimmu.2025.1680896

Figure Lengend Snippet: IDO1 protein knockdown abrogates the pro-tumorigenic phenotype of FTC-133 cells. (A) Immunoblot analysis of IDO1 protein expression in FTC-133 cells transfected with IDO1-specific ( IDO1 siRNA) or negative control siRNAs (n.c. siRNA). Untreated cells were used as a control (ctrl), whereas β-tubulin expression was used as a normalizer. One representative immunoblot of five is shown. The IDO1/β-tubulin protein ratio of scanning densitometry analysis is reported as FC of siRNA-transfected versus respective ctrl cells (1-fold), above the corresponding bands. (B) Kynurenine released by FTC-133 cells treated as in (A) . Results are shown as kynurenine FC of siRNA-transfected versus respective ctrl cells (dotted line, 1-fold). In (A, B) , representative data at 72 h were shown for the ctrl and n.c. siRNA samples. (C) Analysis of the wound closure (black dotted lines) in FTC-133 cells after 48 h of transfection with IDO1 or n.c. siRNAs, over time. Untreated cells were used as ctrl. For each reported time point, one representative image of three is shown (10× magnification; scale bar: 100 µm), and data are reported as wound closure percentage (%) of respective time 0 (time 0 = 0%; dotted line, 50%). (D) Transwell migration assay performed on FTC-133 cells in serum-free medium, after 48 h of transfection with IDO1 or n.c. siRNAs. Untreated cells were used as a ctrl, while untreated cells cultured over serum-free medium were included as a negative control (n.c.). For each condition, a representative field of view is shown (10× magnification; scale bar: 100 µm), and cell migration is reported as percentage (%) of cell-covered area, calculated in each field of view. Data in (A, B, D) and (C) are mean ± SD of independent experiments ( A , B , D : N = 5; C : N = 3). Data in (A-D) were analyzed by one-way or two-way ANOVA followed by post hoc Bonferroni’s test, respectively, comparing IDO1 siRNA versus n.c. siRNA. Statistical significance shown in (A, B) is representative of all comparisons ( IDO1 siRNA at each time point versus n.c. siRNA).

Article Snippet: IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4ETM, Cell Signaling Technology, Danvers, MA, USA), whereas the SHP-2 protein level was detected using a mouse monoclonal anti -SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Knockdown, Western Blot, Expressing, Transfection, Negative Control, Control, Transwell Migration Assay, Cell Culture, Migration

Journal: Frontiers in Immunology

Article Title: Adverse pro-tumorigenic effects of IDO1 catalytic inhibitors mediated by the non-enzymatic function of IDO1 in tumor cells

doi: 10.3389/fimmu.2025.1680896

Figure Lengend Snippet: IDO1 protein degradation abrogates the pro-tumorigenic phenotype of FTC-133 cells. (A) Immunoblot analysis of IDO1 protein expression in FTC-133 cells exposed to iDeg-1 (10 µM) or vehicle alone (ctrl). β-tubulin expression was used as a normalizer. One representative immunoblot of five is shown. The IDO1/β-tubulin protein ratio of scanning densitometry analysis is reported as FC of treated versus respective ctrl cells (1-fold), above the corresponding bands. (B) Kynurenine released by FTC-133 cells treated as in (A) . Results are shown as kynurenine FC of treated versus respective ctrl cells (dotted line, 1-fold). (C) Analysis of the wound closure (black dotted lines) in FTC-133 cells in the presence of LIN (1 µM), iDeg-1 (10 µM), or vehicle alone (ctrl) over time. For each reported time point, one representative image of three is shown (10× magnification; scale bar: 100 µm), and data are reported as wound closure percentage (%) of respective time 0 (time 0 = 0%; dotted line, 50%). (D) Transwell migration assay performed on FTC-133 cells exposed to LIN (1 µM) or iDeg-1 (10 µM) in serum-free medium. Vehicle-treated cells were used as a control, while vehicle-treated cells cultured over serum-free medium were included as negative control (n.c.). For each condition, a representative field of view is shown (10× magnification; scale bar: 400 µm), and cell migration is reported as percentage (%) of cell-covered area, calculated in each field of view. (E) Soft agar colony formation assay in FTC-133 cells exposed to LIN (1 µM), iDeg-1 (10 µM), or vehicle alone once a week. For each condition, the colony size (µm) of 100 representative colonies is reported, whereas one representative image of three is shown (10× magnification; scale bar: 100 µm). Data in (A–E) are mean ± SD of independent experiments ( A , B , D : N = 5; C , E : N = 3). Data in (A–C) and (D, E) were analyzed by two-way or one-way ANOVA followed by post-hoc Bonferroni’s test, respectively. Statistical significance shown in (D) is representative of all comparisons (LIN or iDeg-1 versus ctrl).

Article Snippet: IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4ETM, Cell Signaling Technology, Danvers, MA, USA), whereas the SHP-2 protein level was detected using a mouse monoclonal anti -SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot, Expressing, Transwell Migration Assay, Control, Cell Culture, Negative Control, Migration, Soft Agar Assay

Journal: Frontiers in Immunology

Article Title: Adverse pro-tumorigenic effects of IDO1 catalytic inhibitors mediated by the non-enzymatic function of IDO1 in tumor cells

doi: 10.3389/fimmu.2025.1680896

Figure Lengend Snippet: Dual role of IDO1 in the TME and therapeutic strategies. IDO1 exerts a dual role in the TME through its enzymatic and non-enzymatic functions. Its enzymatic function is mediated by the heme-containing catalytic site in the large domain, which converts tryptophan (Trp) into kynurenine (Kyn). Both Trp depletion and Kyn accumulation foster an immunosuppressive TME that facilitates tumor immune escape (extrinsic effect). The non-enzymatic function is triggered by phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which recruit SHP-2 and activate downstream signaling within tumor cells, thereby promoting tumor growth (intrinsic effect). Conventional IDO1 catalytic inhibitors block only the enzymatic activity and may unexpectedly enhance ITIM-dependent signaling, limiting therapeutic efficacy. By contrast, iDeg-1 induces IDO1 degradation, abolishing both enzymatic and non-enzymatic functions, and thus achieves broader suppression within the TME ( image created with BioRender.com ).

Article Snippet: IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4ETM, Cell Signaling Technology, Danvers, MA, USA), whereas the SHP-2 protein level was detected using a mouse monoclonal anti -SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Phospho-proteomics, Blocking Assay, Activity Assay, Drug discovery