Journal: Molecules

Article Title: Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3- b ]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling

doi: 10.3390/molecules29153515

Figure Lengend Snippet: Compound 5r [ SU1261 ] inhibits FCS-stimulated p100 phosphorylation (Ser866/870) with no impact on TNFα-stimulated IkBα degradation and limited impact on phosphorylation of p65 (Ser536) in U2OS osteosarcoma cells. U2OS cells were grown to near confluency and rendered quiescent by serum deprivation for 24 h. In panel ( A ), cells were then exposed to vehicle (DMSO; 0.15% ( v / v )) or increasing concentrations of 5r [ SU1261 ] (0.3–30 µM) for 1 h prior to treatment with FCS (10% ( v / v )) for 4 h, and phospho-p100 (Ser866/870) was assessed by Western blotting. In panel ( B ), cells were exposed to vehicle (DMSO; 0.15% ( v / v )) or increasing concentrations of 5r [ SU1261 ] (0.3–30 µM) for 1 h prior to treatment with TNFα (10 ng/mL) for 30 min and IkBα degradation, phospho-p65 (Ser536), and p65 expression assessed by Western blotting. The results in panels ( A , B ) are representative of three independent experiments. Normalised data ( n = 3) from semi-quantitative scanning densitometry were plotted relative to ‘agonist plus vehicle’ (FCS plus DMSO), and IC 50 values were established by curve fitting using the Hill equation (see ).

Article Snippet: The antibody-detecting components of the NF-kB pathways used in pharmacodynamic assays were purchased from Cell Signaling Technology (CST) Europe: p-p100 (Ser866/870), p100/p52, IkBα, p-65, p-p65 NF-κB (Ser536), and nucleolin.

Techniques: Western Blot, Expressing

Journal: Molecules

Article Title: Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3- b ]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling

doi: 10.3390/molecules29153515

Figure Lengend Snippet: Compound 6g [ SU1349 ] inhibits FCS-stimulated p100 phosphorylation (Ser866/870) but not TNFα-stimulated IkBα degradation nor phosphorylation of p65 (Ser536) in U2OS osteosarcoma cells. U2OS cells were grown to near confluency and rendered quiescent by serum deprivation for 24 h. In panel ( A ), cells were then exposed to vehicle (DMSO; 0.15% ( v / v )) or increasing concentrations of 6g [ SU1349 ] (0.3–30 µM) for 1 h prior to treatment with FCS (10% ( v / v )) for 4 h, and phospho-p100 (Ser866/870) in whole-cell extracts was assessed by Western blotting. In panel ( B ), cells were exposed to vehicle (DMSO; 0.15% ( v / v )) or increasing concentrations of 6g [ SU1349 ] (0.3–30 µM) for 1 h prior to treatment with TNFα (10 ng/mL) for 30 min, and IkBα degradation, phospho-p65 (Ser536), and p65 expression in whole-cell extracts were assessed by Western blotting. The results in panels ( A , B ) are representative of three independent experiments. Normalised data ( n = 3) from semi-quantitative scanning densitometry were plotted relative to ‘agonist plus vehicle’ (FCS plus DMSO), and IC 50 values were established by curve fitting using the Hill equation (see ).

Article Snippet: The antibody-detecting components of the NF-kB pathways used in pharmacodynamic assays were purchased from Cell Signaling Technology (CST) Europe: p-p100 (Ser866/870), p100/p52, IkBα, p-65, p-p65 NF-κB (Ser536), and nucleolin.

Techniques: Western Blot, Expressing

Journal: Molecules

Article Title: Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3- b ]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling

doi: 10.3390/molecules29153515

Figure Lengend Snippet: Compound 4 inhibits FCS-stimulated p100 phosphorylation (Ser866/870) and TNFα-stimulated IkBα degradation as well as phosphorylation of p65 (Ser536) in U2OS osteosarcoma cells. U2OS cells were grown to near confluency and rendered quiescent by serum deprivation for 24 h. In panel ( A ), cells were then exposed to vehicle (DMSO; 0.15% ( v / v )) or increasing concentrations of 4 (0.3–30 µM) for 1 h prior to treatment with FCS (10% ( v / v )) for 4 h, and phospho-p100 (Ser866/870) in whole-cell extracts was assessed by Western blotting. In panel ( B ), cells were exposed to vehicle (DMSO; 0.15% ( v / v )) or increasing concentrations of 4 (0.3–30 µM) for 1 h prior to treatment with TNFα (10 ng/mL) for 30 min, and IkBα degradation, phospho-p65 (Ser536), and p65 expression in whole-cell extracts were assessed by Western blotting. The results in panels ( A , B ) are representative of three independent experiments. I Normalised data ( n = 3) from semi-quantitative scanning densitometry were plotted relative to ‘agonist plus vehicle’ (FCS plus DMSO), and IC 50 values were established by curve fitting using the Hill equation (see ).

Article Snippet: The antibody-detecting components of the NF-kB pathways used in pharmacodynamic assays were purchased from Cell Signaling Technology (CST) Europe: p-p100 (Ser866/870), p100/p52, IkBα, p-65, p-p65 NF-κB (Ser536), and nucleolin.

Techniques: Western Blot, Expressing

Journal: Molecules

Article Title: Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3- b ]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling

doi: 10.3390/molecules29153515

Figure Lengend Snippet: Compound 5r [ SU1261 ] inhibits LTα 1 β 2 -stimulated p100 phosphorylation (Ser866/870) but not TNFα-stimulated IkBα degradation, phosphorylation of p65 (Ser536), nor phosphorylation of p105 (Ser932) in PC-3M prostate cancer cells. PC-3M cells were grown to near confluency and rendered quiescent by serum deprivation for 24 h. In panel ( A ), cells were then exposed to vehicle (DMSO; 0.05% ( v / v )) or increasing concentrations of 5r [ SU1261 ] (0.1–10 µM) for 1 h prior to treatment with LTα 1 β 2 (15 ng/mL) for 4 h, and phospho-p100 (Ser866/870) in whole-cell extracts was assessed by Western blotting. In panel ( B ), cells were exposed to vehicle (DMSO; 0.05% ( v / v )) or increasing concentrations of 5r [ SU1261 ] (0.1–10 µM) for 1 h prior to treatment with TNFα (10 ng/mL) for 30 min, and IkBα degradation, phospho-p65 (Ser536), phospho-p105 (Ser932), and p65 expression in whole-cell extracts were assessed by Western blotting. The results in panels ( A , B ) are representative of three independent experiments. Normalised data ( n = 3) from semi-quantitative scanning densitometry were plotted relative to ‘agonist plus vehicle’ (LTα 1 β 2 plus DMSO), and IC 50 values were established by curve fitting using the Hill equation (see ).

Article Snippet: The antibody-detecting components of the NF-kB pathways used in pharmacodynamic assays were purchased from Cell Signaling Technology (CST) Europe: p-p100 (Ser866/870), p100/p52, IkBα, p-65, p-p65 NF-κB (Ser536), and nucleolin.

Techniques: Western Blot, Expressing

Journal: Molecules

Article Title: Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3- b ]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling

doi: 10.3390/molecules29153515

Figure Lengend Snippet: Compound 6g [ SU1349 ] inhibits LTα 1 β 2 -stimulated p100 phosphorylation (Ser866/870) and p52/Rel B nuclear translocation but not TNFα-stimulated IkBα degradation, phosphorylation of p65 (Ser536), nor phosphorylation of p105 (Ser932) in PC-3M prostate cancer cells. PC-3M cells were grown to near confluency and rendered quiescent by serum deprivation for 24 h. In panel ( A ), cells were then exposed to vehicle (DMSO; 0.05% ( v / v )) or increasing concentrations of 6g [ SU1349 ] (0.01–3 µM) for 1 h prior to treatment with LTα 1 β 2 (15 ng/mL) for 4 h, and phospho-p100 (Ser866/870) in whole-cell extracts was assessed by Western blotting. In panel ( B ), cells were exposed to vehicle (DMSO; 0.05% ( v / v )) or increasing concentrations of 6g [ SU1349 ] (0.3–30 µM) for 1 h prior to treatment with LTα 1 β 2 (15 ng/mL) for 4 h, and p52/RelB in crude nuclear extracts was assessed by Western blotting. The results in panels ( A , B ) are representative of three independent experiments. Normalised data ( n = 3) from semi-quantitative scanning densitometry were plotted relative to ‘agonist plus vehicle’ (LTα 1 β 2 plus DMSO), and an IC 50 value was established by curve fitting using the Hill equation (see ). In panel ( C ), cells were exposed to vehicle (DMSO; 0.05% ( v / v )) or increasing concentrations of 6g [ SU1349 ] (0.1–10 µM) for 1 h prior to treatment with TNFα (10 ng/mL) for 30 min, and IkBα degradation, phospho-p65 (Ser536), phospho-p105 (Ser932), and p65 expression in whole-cell extracts was assessed by Western blotting.

Article Snippet: The antibody-detecting components of the NF-kB pathways used in pharmacodynamic assays were purchased from Cell Signaling Technology (CST) Europe: p-p100 (Ser866/870), p100/p52, IkBα, p-65, p-p65 NF-κB (Ser536), and nucleolin.

Techniques: Translocation Assay, Western Blot, Expressing

Journal: Communications Biology

Article Title: Decidual natural killer cells dysfunction is caused by IDO downregulation in dMDSCs with Toxoplasma gondii infection

doi: 10.1038/s42003-024-06365-5

Figure Lengend Snippet: a The expression of p52, RelB and IDO in purified human dMDSCs of uninfected and infected groups were examined by qPCR (data represent the mean ± SD from five independent experiments). b The protein levels of IDO, STAT3, p-STAT3 (Y705), IKKα, p-IKKα, p-p100 in purified human dMDSCs, p52 and RelB in nucleus of dMDSCs of uninfected and infected groups were determined by western blot. c The statistical analysis of IDO, p-STAT3 (Y705), p-IKKα, p-p100, p52 and RelB in protein levels from infected groups and uninfected human dMDSCs by western blot. (data represent the mean ± SD from three independent experiments). d The expression levels of p-STAT3 in mouse dMDSCs were examined by flow cytometry in uninfected ( n = 7) and infected groups ( n = 8) and the results were analyzed by statistical analysis. Data are presented as the mean ± SD, * P < 0.05, ** P < 0.01, human samples in each group assayed individually by the paired t -test and mice samples in each group assayed individually by the unpaired t -test. MFI: mean fluorescence intensity.

Article Snippet: The primary antibodies used included STAT3 (Proteintech, China, 10253), p-STAT3 (CST, UK, 9145S), IDO (Abcam, UK, ab76157), IKKα (Wanleibio, China, WL00053), p- IKKα (ABclonal, China, AP0506), p-p100 (ABclonal, China, AP1367), p52 (Proteintech, China, 15503), RelB (Proteintech, China, 66947), SOCS3 (Wanleibio, China, WL01364), GAPDH (Proteintech, China, 10494), LaminB (Wanleibio, China, WL01775), AhR (Wanleibio, China, WL02657), SP1 (Proteintech, China, 21962), TGF-β (Bioss, China, bs-0086R) and IL-10 (Wanleibio, China, WL03088).

Techniques: Expressing, Purification, Infection, Western Blot, Flow Cytometry, Fluorescence

Journal: Communications Biology

Article Title: Decidual natural killer cells dysfunction is caused by IDO downregulation in dMDSCs with Toxoplasma gondii infection

doi: 10.1038/s42003-024-06365-5

Figure Lengend Snippet: a The mRNA levels of IDO in human dMDSCs of infected group, infected with static treated group and infected with SN52 treated group were detected by qPCR (data represent the mean ± SD from three independent experiments by one-way ANOVA). b The expression changes of STAT3, p-STAT3 (Y705), IKKα, p-IKKα, p-p100, IDO and the expression of p52 and RelB of nucleus in purifed human dMDSCs of the uninfected, infected and infected with static treated groups were determined by western blot (data represent the mean ± SD from three independent experiments by one-way ANOVA). c The expression changes of IDO in human dMDSCs and the expression of p52 and RelB in nucleus of dMDSCs from the infected and infected with SN52 treated groups were determined by western blot (data represent the mean ± SD from three independent experiments by the paired t -test). d The IDO expression in mouse dMDSCs of infected and infected with JSI-124 groups were examined by flow cytometry ( n = 6). Data are presented as the mean ± SD, * P < 0.05, ** P < 0.01, unpaired t -test. MFI mean fluorescence intensity.

Article Snippet: The primary antibodies used included STAT3 (Proteintech, China, 10253), p-STAT3 (CST, UK, 9145S), IDO (Abcam, UK, ab76157), IKKα (Wanleibio, China, WL00053), p- IKKα (ABclonal, China, AP0506), p-p100 (ABclonal, China, AP1367), p52 (Proteintech, China, 15503), RelB (Proteintech, China, 66947), SOCS3 (Wanleibio, China, WL01364), GAPDH (Proteintech, China, 10494), LaminB (Wanleibio, China, WL01775), AhR (Wanleibio, China, WL02657), SP1 (Proteintech, China, 21962), TGF-β (Bioss, China, bs-0086R) and IL-10 (Wanleibio, China, WL03088).

Techniques: Infection, Expressing, Western Blot, Flow Cytometry, Fluorescence