Journal: PLoS ONE

Article Title: NF-κB Inducing Kinase, NIK Mediates Cigarette Smoke/TNFα-Induced Histone Acetylation and Inflammation through Differential Activation of IKKs

doi: 10.1371/journal.pone.0023488

Figure Lengend Snippet: (A) CSE and TNFα increased the levels of NIK and phosphorylation of IKKα in H292 cells. H292 cells were treated with CSE (1%) for 1 hr or indicated time points. Whole cell extracts (30 µg) were loaded per well on SDS-6.5% polyacrylamide gel, transferred onto nitrocellulose membranes and probed with NIK, p-IKKα and IKKα antibodies. The levels of NIK and p-IKKα are significantly increased in response to CSE in a time-dependent manner. (B) For immunocytochemistry, cells were fixed, and the expression of NIK was determined by immunofluorescence. NIK is shown in red and nuclear DNA (DAPI nuclear staining) in blue. The images were taken by magnification (×400), and selected a representative cellular morphology from three separate experiments. The group without primary antibody was used as a negative control (N.C.). (C) CSE and TNFα increased the levels of NIK and phosphorylation of IKKα in NHBE cells. NHBEs were treated with CSE (0.5%) for indicated time points. (D) The levels of NIK, p-IKKα, and IKKα were increased in CS-exposed mouse lung. Gel pictures shown are representative of at least three separate experiments. After densitometry analysis, the values were normalized against the loading control, actin. Data shown as mean ± S.E. (n = 3–6 per group). *, p<0.05; **, p<0.01; ***, p<0.001, significant compared with control or air-exposed group.

Article Snippet: FITC conjugated anti-mouse and Alexa Fluor 594 anti-rabbit secondary antibodies for 1 hr in dark, and then the slides were rinsed in PBS and mounted with anti-fade DAPI fluoromount (Southern Biotech), and viewed under the Nikon TE2000-E microscope (Nikon, Tokyo, Japan).

Techniques: Phospho-proteomics, Immunocytochemistry, Expressing, Immunofluorescence, Staining, Negative Control, Control

Journal: PLoS ONE

Article Title: NF-κB Inducing Kinase, NIK Mediates Cigarette Smoke/TNFα-Induced Histone Acetylation and Inflammation through Differential Activation of IKKs

doi: 10.1371/journal.pone.0023488

Figure Lengend Snippet: (A) H292 cells were transiently transfected with NIK siRNA then treated with CSE and TNFα for 1 hr. NIK expression in H292 cells treated with NIK siRNA and scrambled control siRNA (scRNA) were measured by immunoblotting. Whole cell extracts (30 µg) were loaded per well on SDS-10% polyacrylamide gel, transferred onto nitrocellulose membrane. Knockdown of NIK showed a decreased level of NIK in H292 cells in response to CSE. (B) CSE and TNFα increased post-translational modifications of IKKα, RelA/p65 (K310), and histone H3 (S10/K9), and the cells lacking NIK showed decreased levels of phosphorylated IKKα, acetylated RelA/p65, and phospho-acetylated histone H3. (C) For immunocytochemistry, phospho-acetylated histone H3 (S10/K9) is shown in red, NIK is shown in green, and nuclear DNA (DAPI nuclear staining) in blue. The fluorescence intensity values in the non-siRNA control were stated as 100% and the other values were normalized, accordingly. The images were taken by magnification (×200), shown is a representative cellular morphology from three separate experiments. After densitometry analysis, the values were normalized against the loading control, actin and histone H3, respectively. Data shown as mean ± S.E. (n = 3–6 per group). *, p<0.05; **, p<0.01, ***, p<0.001, significant compared with control group; #, p<0.05, ##, p<0.01, ###, p<0.001, significant compared with CSE-exposed group.

Article Snippet: FITC conjugated anti-mouse and Alexa Fluor 594 anti-rabbit secondary antibodies for 1 hr in dark, and then the slides were rinsed in PBS and mounted with anti-fade DAPI fluoromount (Southern Biotech), and viewed under the Nikon TE2000-E microscope (Nikon, Tokyo, Japan).

Techniques: Transfection, Expressing, Control, Western Blot, Membrane, Knockdown, Immunocytochemistry, Staining, Fluorescence

Journal: PLoS ONE

Article Title: NF-κB Inducing Kinase, NIK Mediates Cigarette Smoke/TNFα-Induced Histone Acetylation and Inflammation through Differential Activation of IKKs

doi: 10.1371/journal.pone.0023488

Figure Lengend Snippet: (A) H292 cells were transiently transfected with NIK dominant negative plasmid (DN NIK) and then treated with CSE and TNFα for 1 hr. The NIK mutant transfected cells reduced the enhancement of phosphorylated IKKα, acetylated RelA/p65, and phospho-acetylated histone H3 in response to CSE. (B) For immunocytochemistry, phospho-acetylated histone H3 (S10/K9) is shown in red and DNA (DAPI nuclear staining) in blue. The fluorescence intensity values in the non transfection control were stated as 100% and the other values were normalized, accordingly. The images were taken by magnification (×200), shown is a representative cellular morphology from three separate experiments. After densitometry analysis, the values were normalized against the loading control, actin and histone H3, respectively. Data shown as mean ± S.E. (n = 3–6 per group). *, p<0.05; **, p<0.01, ***, p<0.001, significant compared with control group; #, p<0.05, ###, p<0.001, significant compared with CSE-exposed group.

Article Snippet: FITC conjugated anti-mouse and Alexa Fluor 594 anti-rabbit secondary antibodies for 1 hr in dark, and then the slides were rinsed in PBS and mounted with anti-fade DAPI fluoromount (Southern Biotech), and viewed under the Nikon TE2000-E microscope (Nikon, Tokyo, Japan).

Techniques: Transfection, Dominant Negative Mutation, Plasmid Preparation, Mutagenesis, Immunocytochemistry, Staining, Fluorescence, Control