p-iκb kinase (ikk)-α/β (ser176/180  (Cell Signaling Technology Inc)

Journal: Drug Design, Development and Therapy

Article Title: Genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor κB and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

doi: 10.2147/DDDT.S52354

Figure Lengend Snippet: ( A and B ) Effects of genistein (Gen) on nuclear factor (NF)-κB activation in MH7A cells. Notes: ( A ) MH7A cells were pretreated with Gen or not for 2 hours, and then incubated with tumor necrosis factor (TNF)-α for the indicated times. Phosphorylated IκB kinase (p-IKK)-α/β, p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. ( B ) After being pretreated with Gen or not for 2 hours, MH7A cells were then incubated with TNF-α for 15 minutes. The nuclei translocation of NF-κB p65 was assessed by confocal fluorescence microscopy. Cells were immunostained with NF-κB p65 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Results representative of three independent experiments. Bar 10 μm. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Antibodies against phosphorylated (p)-Akt (Ser473), p-IκB kinase (IKK)-α/β (Ser176/180), p-IκBα (Ser32), NF-κB p65, p-NF-κB p65 (Ser536), and p-AMPK (Thr172) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Incubation, Western Blot, Translocation Assay, Fluorescence, Microscopy, Staining

Journal: Drug Design, Development and Therapy

Article Title: Genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor κB and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

doi: 10.2147/DDDT.S52354

Figure Lengend Snippet: ( A–E ) Involvement of Akt in nuclear factor (NF)-κB activation by tumor necrosis factor (TNF)-α. Notes: ( A ) MH7A cells were pretreated with genistein (Gen) or LY294002 for 2 hours, and then incubated with TNF-α for the indicated times. Phosphorylated (p)-Akt levels were determined by Western blot analysis. ( B ) After being pretreated with LY294002 for 2 hours, MH7A cells were then incubated with TNF-α for 15 minutes. p-IκB kinase (p-IKK)-α/β, p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. ( C–E ) Interleukin (IL)-1β, IL-6,and IL-8 production was determined in MH7A cells treated with TNF-α in the presence or absence of LY294002 for 24 hours by enzyme-linked immunosorbent assay. * P <0.05 and ** P <0.01 vsersus group with TNF-α treatment alone. The data are presented as the means ± standard deviation from three independent experiments. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Antibodies against phosphorylated (p)-Akt (Ser473), p-IκB kinase (IKK)-α/β (Ser176/180), p-IκBα (Ser32), NF-κB p65, p-NF-κB p65 (Ser536), and p-AMPK (Thr172) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: Genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor κB and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

doi: 10.2147/DDDT.S52354

Figure Lengend Snippet: ( A–E ) The role of reactive oxygen species (ROS) in tumor necrosis factor (TNF)-α-mediated inflammatory responses. Notes: ( A ) MH7A cells were stimulated with TNF-α (10 ng/mL) for 15 minutes in the presence of genistein (Gen; 20 μM) or N -acetyl-L-cysteine (NAC; 10 mM). 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate was used to determine the generation of intracellular ROS. Intracellular ROS levels were examined by fluorescence microscopy. ( B–D ) The levels of interleukin (IL)-1β, IL-6, and IL-8 were determined in cells treated with TNF-α alone or with NAC for 24 hours by enzymelinked immunosorbent assay. ( E ) MH7A cells pretreated with NAC for 2 hours before incubating with TNF-α for 15 minutes. Phosphorylated IκB kinase (p-IKK)-α/β, p-IκBα, and p-nuclear factor κB p65 levels were determined by Western blot analysis. * P <0.05 and ** P <0.01 versus group with TNF-α treatment alone. The data are presented as the means ± standard deviation from three independent experiments. Bar 100 μm. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Antibodies against phosphorylated (p)-Akt (Ser473), p-IκB kinase (IKK)-α/β (Ser176/180), p-IκBα (Ser32), NF-κB p65, p-NF-κB p65 (Ser536), and p-AMPK (Thr172) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Fluorescence, Microscopy, Western Blot, Standard Deviation

Journal: Drug Design, Development and Therapy

Article Title: Genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor κB and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

doi: 10.2147/DDDT.S52354

Figure Lengend Snippet: Schematic model illustrating the potential pathway associated with genistein’s inhibition of tumor necrosis factor (TNF)-α-induced inflammation. Notes: Genistein suppresses TNF-α-induced proinflammatory cytokine production through 1) reducing intracellular reactive oxygen species (ROS) accumulation triggered by TNF-α, which represses Akt/nuclear factor (NF)-κB signaling, and 2) promoting adenosine monophosphate-activated protein kinase (AMPK) activation. Abbreviations: IKK, IκB kinase; PI3K, phosphoinositide-3 kinase; TNFR, tumor necrosis factor receptor.

Article Snippet: Antibodies against phosphorylated (p)-Akt (Ser473), p-IκB kinase (IKK)-α/β (Ser176/180), p-IκBα (Ser32), NF-κB p65, p-NF-κB p65 (Ser536), and p-AMPK (Thr172) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activation Assay