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pcmv3 c flag ido1  (Sino Biological)


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    Structured Review

    Sino Biological pcmv3 c flag ido1
    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 <t>IDO1</t> 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.
    Pcmv3 C Flag Ido1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/flag-ido1+vector/pCMV3-C-FLAG+Negative+Control+Vector/pmc08902613-252-1-3
    Average 94 stars, based on 55 article reviews
    pcmv3 c flag ido1 - by Bioz Stars, 2026-10
    94/100 stars

    Images

    1) Product Images from "Inhibition of the Na + /K + -ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation"

    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

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.101707

    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.
    Figure Legend 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.

    Techniques Used: Expressing

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Titration, Western Blot, Binding Assay

    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.
    Figure Legend 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.

    Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    Related Articles

    Over Expression:

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    Article Title: Artemether and Euphorbia factor L9 suppress kynurenine production through distinct effects on tryptophan metabolism
    Article Snippet: .. IDO1 overexpression was achieved using a pCMV3-C-Flag-IDO1 ( NM_002164.4 ) (SinoBiological, HG11650-CF) plasmid, while a pCMV3-C-Flag-NCV (SinoBiological, CV012) empty vector was used as a negative control. .. Transfections were carried out using the jetPRIME® DNA /siRNA transfection kit (SARTORIUS, 101000015).

    Article Title: Artemether and Euphorbia Factor L9 suppress kynurenine production through distinct effects on Tryptophan metabolism.
    Article Snippet: .. IDO1 overexpression was achieved using a pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) plasmid, while a pCMV3-C-Flag-NCV (SinoBiological, CV012) empty vector was used as a negative control. .. Transfections were carried out using the jetPRIME® DNA / siRNA transfection kit (SARTORIUS, 101000015).

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer.
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Mutagenesis:

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer.
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Clone Assay:

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer.
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Plasmid Preparation:

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Article Title: Artemether and Euphorbia factor L9 suppress kynurenine production through distinct effects on tryptophan metabolism
    Article Snippet: .. IDO1 overexpression was achieved using a pCMV3-C-Flag-IDO1 ( NM_002164.4 ) (SinoBiological, HG11650-CF) plasmid, while a pCMV3-C-Flag-NCV (SinoBiological, CV012) empty vector was used as a negative control. .. Transfections were carried out using the jetPRIME® DNA /siRNA transfection kit (SARTORIUS, 101000015).

    Article Title: Artemether and Euphorbia Factor L9 suppress kynurenine production through distinct effects on Tryptophan metabolism.
    Article Snippet: .. IDO1 overexpression was achieved using a pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) plasmid, while a pCMV3-C-Flag-NCV (SinoBiological, CV012) empty vector was used as a negative control. .. Transfections were carried out using the jetPRIME® DNA / siRNA transfection kit (SARTORIUS, 101000015).

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer.
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression
    Article Snippet: For immunofluorescence, anti-LC3B (Cell Signaling Technology Cat# 3868, RRID:AB_2137707), anti-mouse IgG Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 4409, RRID:AB_1904022), anti-rabbit Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 19581, RRID:AB_2943221), anti-rabbit IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4412, RRID:AB_1904025), anti-mouse IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4523, RRID:AB_836889) were purchased from Cell Signaling Technology. .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression.
    Article Snippet: .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http:// n2t.net/addgene:12259; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).

    Transfection:

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Article Title: Ginsenoside Rh2 as a novel PIN1 inhibitor disrupting the cancer stem cell-like phenotype in non-small cell lung cancer.
    Article Snippet: .. For the establishment of protein overexpression cell lines, the CDS sequences of PIN1, PGK1, and PGK1 S203A mutant were cloned into the pCMV3-C-FLAG vector (SinoBiological, China), and then introduced into recipient cells using Lipofectamine 2000 transfection reagent (Thermo, USA). .. Exponentially growing cells were seeded at a density of 1,000 cells/mL in serum-free DMEM/F12 medium (Laisi Biotechnology, Shanghai, China), supplemented with 2% B-27 (Yeasen Biotechnology, Shanghai, China), 1% N2 (Yeasen Biotechnology, Shanghai, China), 0.2% epidermal growth factor (EGF) (20 ng/mL) (R&D Systems, USA), and 10 ng/mL basic fibroblast growth factor (PeproTech, USA).

    Negative Control:

    Article Title: Artemether and Euphorbia factor L9 suppress kynurenine production through distinct effects on tryptophan metabolism
    Article Snippet: .. IDO1 overexpression was achieved using a pCMV3-C-Flag-IDO1 ( NM_002164.4 ) (SinoBiological, HG11650-CF) plasmid, while a pCMV3-C-Flag-NCV (SinoBiological, CV012) empty vector was used as a negative control. .. Transfections were carried out using the jetPRIME® DNA /siRNA transfection kit (SARTORIUS, 101000015).

    Article Title: Artemether and Euphorbia Factor L9 suppress kynurenine production through distinct effects on Tryptophan metabolism.
    Article Snippet: .. IDO1 overexpression was achieved using a pCMV3-C-Flag-IDO1 (NM_002164.4) (SinoBiological, HG11650-CF) plasmid, while a pCMV3-C-Flag-NCV (SinoBiological, CV012) empty vector was used as a negative control. .. Transfections were carried out using the jetPRIME® DNA / siRNA transfection kit (SARTORIUS, 101000015).

    Ubiquitin Proteomics:

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression
    Article Snippet: For immunofluorescence, anti-LC3B (Cell Signaling Technology Cat# 3868, RRID:AB_2137707), anti-mouse IgG Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 4409, RRID:AB_1904022), anti-rabbit Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 19581, RRID:AB_2943221), anti-rabbit IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4412, RRID:AB_1904025), anti-mouse IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4523, RRID:AB_836889) were purchased from Cell Signaling Technology. .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression.
    Article Snippet: .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http:// n2t.net/addgene:12259; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).

    CRISPR:

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression
    Article Snippet: For immunofluorescence, anti-LC3B (Cell Signaling Technology Cat# 3868, RRID:AB_2137707), anti-mouse IgG Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 4409, RRID:AB_1904022), anti-rabbit Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 19581, RRID:AB_2943221), anti-rabbit IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4412, RRID:AB_1904025), anti-mouse IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4523, RRID:AB_836889) were purchased from Cell Signaling Technology. .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression.
    Article Snippet: .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http:// n2t.net/addgene:12259; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).

    Knock-Out:

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression
    Article Snippet: For immunofluorescence, anti-LC3B (Cell Signaling Technology Cat# 3868, RRID:AB_2137707), anti-mouse IgG Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 4409, RRID:AB_1904022), anti-rabbit Alexa Fluor® 555 conjugate (Cell Signaling Technology Cat# 19581, RRID:AB_2943221), anti-rabbit IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4412, RRID:AB_1904025), anti-mouse IgG Alexa Fluor® 488 conjugate (Cell Signaling Technology Cat# 4523, RRID:AB_836889) were purchased from Cell Signaling Technology. .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).

    Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression.
    Article Snippet: .. pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http:// n2t.net/addgene:12259; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological. .. HEK293TN cells (BioCat cat. # LV900A-1-GVO-SBI, RRID: CVCL_UL49) were grown in DMEM medium containing 10% inactivated fetal bovine serum (FBS) (GibcoTM Cat# A3160501).



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    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 <t>IDO1</t> 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.
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    Image Search Results


    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).

    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

    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).

    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

    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).

    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

    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).

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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