ido1 plasmid vector  (Sino Biological)

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation

doi: 10.3389/fimmu.2022.832263

Figure Lengend Snippet: CLL cells up-regulate IDO1 in response to stimuli that mimic microenvironmental signals. (A) Purified leukemic CD19 + cells from CLL patients were serum starved for 1 h. Then the basal level of IDO1 was inspected by Western blot (n = 8) and illustrated by a bar diagram. (B) Flow cytometric histograms represent a high IDO1 level in CD19 + cells treated for 24 h with IFN-γ compared to the untreated control sample and isotype sample. (C) Bar diagram represents IDO1 expression in CD19 + CLL measured by real-time PCR. Samples were treated for 4 h with one of microenvironment stimuli individually (Student paired t test, *p < 0.05, **p < 0.01; n = 5). (D) Immunoblots represent IDO1 protein induction after 24 h of stimulation with the abovementioned factors. Histograms below represent the densitometric quantifications (Student paired t test, *p < 0.05, **p < 0.01; n = 5).

Article Snippet: Briefly, 5 × 10 6 CD19 + CLL cells were transfected with 10 μg of IDO1 plasmid vector (Cat# HG11650-NF) or the corresponding empty vector pCMV3-Negative Control (Cat# CV020) (all from Sino Biological Inc., Shanghai, China).

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

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation

doi: 10.3389/fimmu.2022.832263

Figure Lengend Snippet: IDO1 protein induction and activity can be regulated by IFN-γ in CLL. (A) Purified CLL cells were pretreated with two doses (0.1–1 μM) of INCB018424 for 1 h prior to IFN-γ stimulation for 24 h. Immunoblots depict IDO1, tSTAT1, and pSTAT1 protein levels in a representative case. Histograms on the right represent the densitometric quantifications of IDO1 and pSTAT1/tSTAT1 in 5 CLL samples (Student paired t test, *p < 0.05; n = 5). (B) Immunofluorescence staining shows the ability of INCB018424 1 μM to reduce IDO1 expression induced by IFN-γ. Scale bar is 20 µm. Box plots summarize the fluorescent levels in DMSO, IFN-γ, or IFN-γ + INCB018424-treated CLL cells (Student paired t test, *p < 0.05; n = 9). (C) Conditioned media were collected after INCB018424 pretreatment (or not) and IFN-γ incubation for 24 h. Dot plots on the left represent the mean of the Kyn concentration measured by LC-MS/MS analytical technique in 5 separated experiments (Student paired t test, *p < 0.05, **p < 0.01; n = 5). The central dot plots represent the mean of the Trp concentration (Student paired t test, *p < 0.05, ***p < 0.001; n = 5). The resultant [Kyn]/[Trp] ratio calculated is depicted in the right dot plot (Student paired t test, *p < 0.05, **p < 0.01; n = 5).

Article Snippet: Briefly, 5 × 10 6 CD19 + CLL cells were transfected with 10 μg of IDO1 plasmid vector (Cat# HG11650-NF) or the corresponding empty vector pCMV3-Negative Control (Cat# CV020) (all from Sino Biological Inc., Shanghai, China).

Techniques: Activity Assay, Purification, Western Blot, Immunofluorescence, Staining, Expressing, Incubation, Concentration Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation

doi: 10.3389/fimmu.2022.832263

Figure Lengend Snippet: CLL cell survival is promoted by IDO1 overexpression. (A) CLL cells were transfected with an IDO1-expressing vector or an empty vector. After 24 h from transfection, the IDO1 protein level was measured by Western blot. Bar diagrams represent the densitometric quantifications of IDO1 (Student paired t test, *p < 0.05; n = 5). (B) Conditioned media were collected 24 h post transfection with the IDO1 vector or empty vector. Dot plots on the left represent the mean of the Kyn concentration measured by HPLC in 8 separated experiments (Student paired t test, **p < 0.01; n = 8). The central dot plots represent the mean of the Trp concentration (Student paired t test, *p < 0.05; n = 8). The resultant [Kyn]/[Trp] ratio calculated is depicted in the right dot plot (Student paired t test, **p < 0.01; n = 8). (C) Box plots represent the percentage of viable CLL cells after 24 h of transfection with IDO1-expressing vector or empty vector (Student paired t test, *p < 0.05; n = 5). (D) Box plots represent the percentage of CLL cell viability measured after 48 h of stimulation with Kyn 100 µM (Student paired t test, **p < 0.01; n = 6).

Article Snippet: Briefly, 5 × 10 6 CD19 + CLL cells were transfected with 10 μg of IDO1 plasmid vector (Cat# HG11650-NF) or the corresponding empty vector pCMV3-Negative Control (Cat# CV020) (all from Sino Biological Inc., Shanghai, China).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation

doi: 10.3389/fimmu.2022.832263

Figure Lengend Snippet: The activity of IDO1/Kyn/AHR axis impairs CLL cells response to ABT-199. (A) Histograms show comparison on viability rate of CLL cells treated with 1 nM ABT-199 for 5 h after overnight stimulation with 100 µM Kyn (repeated-measure two-way ANOVA with Kyn and CH-223191 treatment groups as the two factors; n = 11). (B) CLL cells were pretreated for 90 min with 10 µM CH-223191 and cultured overnight with Kyn. Then, 1 nM ABT-199 was added to culture for 5 h prior to cell viability assessment. Histograms illustrate the synergistic effect of CH-223191 with ABT-199 in CLL cells (repeated-measure three-way ANOVA with Kyn, ABT-199, and CH-223191 treatment groups as the three factors; n = 7). (C) CD19+ cells were cultured overnight with 100 µM Kyn, and then 100 or 300 nM AMG-176 was added to culture for 5 h. Cell viability was measured and depicted in histograms (repeated-measure two-way ANOVA with Kyn and AMG-176 treatment groups as the two factors; n = 12).

Article Snippet: Briefly, 5 × 10 6 CD19 + CLL cells were transfected with 10 μg of IDO1 plasmid vector (Cat# HG11650-NF) or the corresponding empty vector pCMV3-Negative Control (Cat# CV020) (all from Sino Biological Inc., Shanghai, China).

Techniques: Activity Assay, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of the Na + /K + -ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

doi: 10.1016/j.jbc.2022.101707

Figure Lengend Snippet: Ion channel targeting small-molecule screen of kynurenine production. A , experimental setup of the ion channel targeting small molecule screen. Cell lines were treated with ion channel targeting small molecules for 24 h, then stimulated with TNF/IFN-γ for 24 h to induce IDO1 expression before assaying kynurenine production. B , kynurenine assay scheme. C , MDA-MB-231 screen results. Data were normalized to their respective DMSO control, 0.05% or 1% DMSO. ∗∗∗∗ p < 0.0001, one-way ANOVA Tukey’s post-hoc. D , A549 screen results. Data were normalized to their respective DMSO control, 0.05% or 1% DMSO. ∗∗∗∗ p < 0.0001, one-way ANOVA Tukey’s post-hoc. IDO1, indoleamine-pyrrole 2′,3′-dioxygenase 1.

Article Snippet: A pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) and a pCMV3-C-Flag empty vector (CV012) were purchased from SinoBiological.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of the Na + /K + -ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

doi: 10.1016/j.jbc.2022.101707

Figure Lengend Snippet: Cardiac glycosides ouabain and digoxin inhibit kynurenine production and IDO1 protein expression in MDA-MB-231 and A549 cells. A , dose-response curve for ouabain and digoxin treatment of TNF/IFN-ɣ stimulated MDA-MB-231 cells. IC 50 ouabain = 89 nM, 95% confidence interval 70 to 112 nM. Apparent IC 50 digoxin ∼164 nM, n = 3. B , dose–response curve for ouabain and digoxin treatment of TNF/IFN-ɣ stimulated A549 cells. IC 50 ouabain = 17 nM, 95% confidence interval 14 to 20 nM. IC 50 digoxin 40 nM, 95% confidence interval 35 to 46 nM, n = 3. C , representative SBFI data from three independent experiments measuring intracellular sodium in response to 24-h cardiac glycoside or 0.05% DMSO control treatment of TNF/IFN-ɣ-stimulated MDA-MB-231 cells. ∗∗∗ p < 0.001, ∗∗ p < 0.01, One-way ANOVA, Tukey’s multiple comparisons. D , ouabain titration inhibits kynurenine production (3 independent experiments, •) and increases intracellular sodium ([Na + ] i (2 independent experiments, ▪) in TNF/IFN-ɣ stimulated MDA-MB-231 cells treated with the indicated concentrations of ouabain. Data were fitted using nonlinear regression by least squares fit. Pearson Correlation r 2 = 0.9628, p < 0.05. E , representative Western blots of IDO1 protein expression in TNF/IFN-ɣ-stimulated MDA-MB-231 and A549 cells pre-treated with ouabain or 0.05% DMSO. F , IDO1 and GAPDH protein expression in MDA-MB-231 ( top ) or A549 ( bottom ) cells pretreated with ouabain, digoxin, or 0.05% DMSO control for 36 h, then TNF/IFN-ɣ stimulated for 24 h. Ouabain and digoxin treated samples were visualized on two different blots. G , relative IDO1 band intensities determined from Western blots of three independent experiments in which TNF/IFN-ɣ-stimulated MDA-MB-231 cells were pretreated with 0.05% DMSO or 100 nM ouabain. H , IDO1 mRNA levels from three independent experiments in which TNF/IFN-ɣ stimulated MDA-MB-231 cells were pretreated with 0.05% DMSO or 100 nM ouabain. IDO1, indoleamine-pyrrole 2′,3′-dioxygenase 1; SBFI, sodium-binding benzofuran isophthalate.

Article Snippet: A pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) and a pCMV3-C-Flag empty vector (CV012) were purchased from SinoBiological.

Techniques: Expressing, Titration, Western Blot, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of the Na + /K + -ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

doi: 10.1016/j.jbc.2022.101707

Figure Lengend Snippet: IDO1 expression is sufficient to produce kynurenine in MDA-MB-231 cells. A , representative Western blot of IDO1 protein expression in TNF/IFN-ɣ-stimulated MDA-MB-231 cells, pretreated with increasing concentrations of ouabain or 0.05% DMSO, and transfected with an IDO1 targeting siRNA pool or nontargeting control (NTC) siRNA pool. B , kynurenine assay of TNF/IFN-ɣ-stimulated MDA-MB-231 cells, pretreated with increasing concentrations of ouabain or 0.05% DMSO, and transfected with an IDO1 targeting siRNA pool or nontargeting control (NTC) siRNA pool. Three independent experiments. C , representative Western blot of IDO1 protein expression in MDA-MB-231 cells transfected with pIDO1-FLAG or empty vector and pretreated with 0.05% DMSO (ctrl), 100 nM ouabain (OUA), no stimulation (no stim), or TNF/IFN-ɣ. D , kynurenine assay of MDA-MB-231 cells transfected with pIDO1-FLAG or empty vector and pretreated with 0.05% DMSO or 100 nM ouabain or no stimulation or TNF/IFN-ɣ. Three independent experiments. ∗ p < 0.05, one-way ANOVA Tukey’s post-hoc. IDO1, indoleamine-pyrrole 2′,3′-dioxygenase 1.

Article Snippet: A pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) and a pCMV3-C-Flag empty vector (CV012) were purchased from SinoBiological.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of the Na + /K + -ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

doi: 10.1016/j.jbc.2022.101707

Figure Lengend Snippet: ATP1A1 knockdown and cardiac glycoside treatment result in synergistic inhibition of IDO1 expression and activity in MDA-MB-231 cells. A , SBFI intracellular Na + measurements in NTC versus ATP1A1 siRNA transfected cells, n = 3. ∗ p < 0.05, unpaired, two-tailed t test. Inset , representative Western blot of ATP1A1 and GAPDH protein expression in samples transfected with NTC or ATP1A1 siRNA. B , kynurenine assay of untransfected, NTC siRNA, or ATP1A1 siRNA-transfected TNF/IFN-ɣ (24 h) stimulated MDA-MB-231 cells treated with 0.05% DMSO or increasing concentrations of ouabain (50, 100, 200 nM), n = 3. Inset , normalized viability (inhibitor/DMSO control) versus normalized IDO activity (inhibitor/DMSO control) from each of three wells of one kynurenine assay. C , kynurenine assay of untransfected, NTC siRNA, or ATP1A1 siRNA transfected TNF/IFN-ɣ (24 h) stimulated MDA-MB-231 cells treated with 0.05% DMSO or increasing concentrations of digoxin (75, 150, 300 nM), n = 3. Inset , normalized viability (inhibitor/DMSO control) versus normalized IDO activity (inhibitor/DMSO control) from each of three wells of one kynurenine assay. D , representative Western blot of ATP1A1, PD-L1, IDO1, and GAPDH protein expression for experiments in ( B ). The same samples were run and blotted simultaneously on two separate membranes. GAPDH loading control for each blot is noted. E , representative Western blot of ATP1A1, PD-L1, IDO1, and GAPDH protein expression for experiments in ( C ). The same samples were run and blotted simultaneously on two separate membranes. GAPDH loading control for each blot is noted. F , IDO1 mRNA expression for NTC or ATP1A1 siRNA transfected, TNF/IFN-ɣ (24 h) stimulated MDA-MB-231 cells treated with increasing concentrations of ouabain, n = 3. G , IDO1 mRNA expression for NTC or ATP1A1 siRNA transfected, TNF/IFN-ɣ (24 h) stimulated MDA-MB-231 cells treated with increasing concentrations of digoxin, n = 3. IDO1, indoleamine-pyrrole 2′,3′-dioxygenase 1; SBFI, sodium-binding benzofuran isophthalate.

Article Snippet: A pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) and a pCMV3-C-Flag empty vector (CV012) were purchased from SinoBiological.

Techniques: Inhibition, Expressing, Activity Assay, Transfection, Two Tailed Test, Western Blot, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of the Na + /K + -ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation

doi: 10.1016/j.jbc.2022.101707

Figure Lengend Snippet: ATP1A1 knockdown and cardiac glycoside treatment result in synergistic inhibition of IDO1 expression and activity in lung cancer A549 cells. A , kynurenine assay of untransfected, NTC siRNA, or ATP1A1 siRNA transfected TNF/IFN-ɣ (24 h) stimulated A549 cells treated with 0.05% DMSO or increasing concentrations of ouabain (10, 25, 50 nM), n = 3. (Inset normalized viability (inhibitor/DMSO control) versus normalized IDO activity (inhibitor/DMSO control) from each of three wells of one kynurenine assay). B , kynurenine assay of untransfected, NTC siRNA, or ATP1A1 siRNA transfected TNF/IFN-ɣ (24 h) stimulated A549 cells treated with 0.05% DMSO or increasing concentrations of digoxin (25, 50, 100 nM), n = 3. Inset , normalized viability (inhibitor/DMSO control) versus normalized IDO activity (inhibitor/DMSO control) from each of three wells of one kynurenine assay. C , representative Western blot of ATP1A1, PD-L1, IDO1, and GAPDH protein expression for experiments in A . The same samples were run and blotted simultaneously on two separate membranes. GAPDH loading control for each blot is noted. D , representative Western blot of ATP1A1, PD-L1, IDO1, and GAPDH protein expression for experiments in B . The same samples were run and blotted simultaneously on two separate membranes. GAPDH loading control for each blot is noted. The dashed vertical lines in PD-L1 and accompanying GAPDH panels indicate an empty lane that has been spliced out. E , IDO1 mRNA expression for NTC or ATP1A1 siRNA transfected, TNF/IFN-ɣ (24 h) stimulated A549 cells treated with increasing concentrations of ouabain, n = 3. F , IDO1 mRNA expression for NTC or ATP1A1 siRNA transfected, TNF/IFN-ɣ (24 h) stimulated A549 cells treated with increasing concentrations of digoxin, n = 3. IDO1, indoleamine-pyrrole 2′,3′-dioxygenase 1.

Article Snippet: A pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) and a pCMV3-C-Flag empty vector (CV012) were purchased from SinoBiological.

Techniques: Inhibition, Expressing, Activity Assay, Transfection, Western Blot