Journal: Molecular and cellular endocrinology

Article Title: Role of the AMP kinase in cytokine-induced human EndoC-βH1 cell death.

doi: 10.1016/j.mce.2015.07.015

Figure Lengend Snippet: Fig. 5. Inhibition of STAT-1 does not prevent cytokine-induced EndoC-bH1 cell death. (A) EnodC-bH1 cells were treated with control or STAT-1 siRNA. Two days later cells were exposed to IL-1b þ IFN-g overnight and cell death rates were analyzed by flow cytometry. For immunoblot analysis of P-STAT-1 cells were exposed to cytokines for 30 min. Upper panel shows the means ± SEM for 4 experiments and the lower panel shows a 55% knockdown of Phospho-STAT-1 induced by siRNA treatment. The intensities of the P-STAT-1 bands were normalized to total protein loading and transfer, as assessed by amidoblack staining. (B) and (C) EndoC-bH1 cells were pre-exposed to 10 mM epigallocathecin gallate (EGCG) or 50 ng/ml fludarabine (Flud) for 15 min and then cultured for another 18 h in the presence of IL-1b þ IFN-g before analysis of cell death using propidium iodide staining and flow cytometry. Results are means ± SEM for 4 independent observations.

Article Snippet: IKK inhibitor X and epigallocathecin gallate was from Santa Cruz.

Techniques: Inhibition, Control, Cytometry, Western Blot, Knockdown, Staining, Cell Culture

Journal: Biochemical and biophysical research communications

Article Title: IKK regulates the deubiquitinase CYLD at the postsynaptic density.

doi: 10.1016/j.bbrc.2014.06.019

Figure Lengend Snippet: Fig. 2. IKK phosphorylates CYLD at S-418 in vitro. (A) PSD fractions were incubated under different conditions designed to manipulate IKK activity, followed by Western immunoblotting with an antibody specific to CYLD phosphorylated at S- 418 (p-CYLD) (top panels) or an antibody to CYLD (bottom panels). Addition of ATP induced phosphorylation of CYLD at S-418, but inclusion of the IKK inhibitors, IKK16 or tatNEMO reduced CYLD phosphorylation in a dose-dependent manner. (B) Purified CYLD and purified IKKb were incubated in the presence or the absence of ATP, followed by Western immunoblotting as described above. Addition of ATP induced phosphorylation of CYLD at S-418. Two experiments yielded similar results.

Article Snippet: IKK16, N-(4-Pyrrolidin-1-yl-piperidin-1-yl)-[4-(4-benzo[b]thiophen-2-yl-pyrimidin2-ylamino)phenyl]carboxamide hydrochloride, is from Tocris (Ellisville, CO).

Techniques: In Vitro, Incubation, Activity Assay, Western Blot, Phospho-proteomics

Journal: Biochemical and biophysical research communications

Article Title: IKK regulates the deubiquitinase CYLD at the postsynaptic density.

doi: 10.1016/j.bbrc.2014.06.019

Figure Lengend Snippet: Fig. 3. The phosphorylation state of CYLD at the PSD of hippocampal neurons is controlled by IKK under basal conditions. Hippocampal cultures were pre- incubated with or without the IKK inhibitor IKK16 for 20 min, followed by treatment with either NMDA or control medium for 2 min before fixation and immunogold labeling with an antibody specific for CYLD phosphorylated on S-418 (p-CYLD). (A) Electron micrographs of the synaptic region showing immunogold labeling for p-CYLD, seen as dark grains of heterogeneous sizes. On the right panel is shown the zone for measuring amount of labeling at the PSD complex, delineated by two perpendicular lines to the postsynaptic membrane and a parallel dashed line at 120 nm distance from the postsynaptic membrane. NMDA treatment promoted an increase in p-CYLD labels at the PSD. (B and C) The histograms depict the frequency of PSDs either with no label (left) or with labels of varying intensities (right). Immunolabeling intensities were binned into groups from 5 to 65. Under basal conditions (B), the IKK inhibitor IKK16 (gray bars) reduced the labeling for phosphorylated CYLD at the PSD. However, preincubation with the inhibitor had no significant effect on labeling for phosphorylated CYLD following NMDA treatment (C). The histograms represent one experiment out of a total of three experiments with similar results.

Article Snippet: IKK16, N-(4-Pyrrolidin-1-yl-piperidin-1-yl)-[4-(4-benzo[b]thiophen-2-yl-pyrimidin2-ylamino)phenyl]carboxamide hydrochloride, is from Tocris (Ellisville, CO).

Techniques: Phospho-proteomics, Incubation, Control, Labeling, Membrane, Immunolabeling

Journal: Biochemical and biophysical research communications

Article Title: IKK regulates the deubiquitinase CYLD at the postsynaptic density.

doi: 10.1016/j.bbrc.2014.06.019

Figure Lengend Snippet: Fig. 4. IKK regulates DUB activity targeting K63-linked polyubiquitins. (A) Purified CYLD was pre-incubated with purified IKKb in the presence or absence of ATP, followed by incubation with tetrameric K63-linked polyubiquitin chains (Ub4). IKKb was omitted in additional controls (right column). The upper two panels are Western immunoblots with antibodies specific for CYLD phosphorylated at S-418 (p-CYLD) and for CYLD respectively, while the bottom panels shows the lower portions of coomassie blue stained gels containing Ub4 and degradation products. Addition of both IKKb and ATP promotes CYLD phosphorylation (upper panels), with a concomitant increase in the rate of cleavage of Ub4 (bottom panels). Two experiments yielded similar results. (B) PSD fractions were incubated under different conditions designed to manipulate IKK activity, followed by incubation with tetrameric K63-linked polyubiquitin chains (Ub4). CYLD phosphorylation status (top panels) in comparison to total CYLD levels (middle panels) and DUB activity (bottom panels) were monitored as described above. Inclusion of ATP promoted CYLD phosphorylation, and an increase in the rate of Ub4 breakdown. Addition of IKK16 prevented both CYLD phosphorylation and ATP-dependent upregulation of Ub4 breakdown (bottom panels). Three independent experiments yielded similar results.

Article Snippet: IKK16, N-(4-Pyrrolidin-1-yl-piperidin-1-yl)-[4-(4-benzo[b]thiophen-2-yl-pyrimidin2-ylamino)phenyl]carboxamide hydrochloride, is from Tocris (Ellisville, CO).

Techniques: Activity Assay, Incubation, Western Blot, Staining, Phospho-proteomics, Comparison

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A. Recombinant DNA constructs employed to generate both transgenic mice lines. For C-IKKα mice generation, the nuclear localization signal (NLS) was removed from the sequence of the human IKKα cDNA employed. In the construct used for generation of the N-IKKα mice an extra NLS signal was added. WT IKKα; wild type IKKα. B. Western blot of total protein extracts showing IKKα expression in back skin of Control and C-and N-IKKα mice. Actin was used as a loading control. C. Representative example of the K5 staining in back skin section of Control mice. D-E. Expression of exogenous IKKα protein in back skin of 1-month-old mice. Immunostaining with the NB100-56704 anti-IKKα antibody is showed; similar results were obtained with the H00001147-M04 IKKα antibody (not shown). Note the cytoplasmic expression of the transgene in the C-IKKα mice (D). By contrast, it is located in the nuclei of cells in the N-IKKα mice (E). In both types of transgenic mice the exogenous IKKα is expressed in basal keratinocytes (bk), in the outer root sheath of hair follicles (ORS) and in cells surrounding the sebaceous glands (sb). F. Back skin section of Control mice. The NB100-56704 antibody used does not recognize the endogenous IKKα in immunohistochemical assays. G. Endogenous IKKα expression in control mice using the IKKα (sc-7182) antibody. Scale bar: (C) 70 μm; (D-G) 60 μm.

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Recombinant, Construct, Transgenic Assay, Sequencing, Western Blot, Expressing, Control, Staining, Immunostaining, Immunohistochemical staining

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A. Western blot showing the increased expression of IKKα in transgenic mice. B-I. Immunohistochemistry showing the expression of the transgenic protein in N-IKKα and C-IKKa tumors. Staining with NB100-56704 antibody is shown. (B, C) Representative images showing the expression of transgenic IKKα in tumors and adjacent skin of N-IKKα/TgAC mice (B), and C-IKKα/TgAC animals (C). (D, E) Detail showing the nuclear (D) or cytoplasmic (E) localization of the transgenic IKKα in tumors. (F, G) Similar levels of expression of the transgenic IKKα in different N-IKKα tumors. By contrast variable levels of expression of the transgene are observed between different C-IKKα tumors (H, I). t: tumor; s: non-tumoral skin. Scale bar: (B, C) 100μm; (D, E) 80 μm; (F-I) 200 μm.

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Western Blot, Expressing, Transgenic Assay, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A, D, G. P-IKKα expression. P-IKKα/β (Ser 180/Ser 181) antibody is used. B, E, H. Specific staining of human IKKα-using the NB100-56704 antibody. C, F, I. Staining with the sc-7182 antibody that recognizes both human and mouse IKKα. Observe that as expected, in the N-IKKα tumors the signal of this antibody is detected both in cytoplasmic and nuclear localization; by contrast, in the Control tumors the endogenous IKKα is mainly observed in the cytosolic compartment, although some nuclear staining is also observed. In the C-IKKα tumors little nuclear staining is observed. Scale bar: 70μm.

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Staining, Control

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A-F. Representative Western blots analysis of IKKα, P-p65, p65, EGFR, P-EGFR, p100/52, Maspin, c-Myc, E-cadherin and MMP-9 expression in Control, C-IKKα and N-IKKα tumors. Actin and GAPDH were used as loading controls. Western blot of protein extracts from 5 to 8 tumors derived from to 4 to 6 different mice of each genotype were performed. The identification of each tumor and mouse corresponding to every lane is provided in G. Bands of the different immunoblots were quantified by Quantity One software and Image Lab software and normalized with respect to Actin or GAPDH expression. P values were determined by Student's t -test and p values <0.05 (*) were considered significant; **p<0.01. H. Determination of VEGF-A mRNA relative levels in skin of Control, C-IKKα and N-IKKα transgenic mice by qRT-PCR analyses ( P <0,05).

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Western Blot, Expressing, Control, Derivative Assay, Software, Transgenic Assay, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: Downregulation of E-cadherin expression in N-IKKα- and C-IKKα tumors. The CD31 staining (marker of endothelial cells) shows the presence of dilated and leaky blood vessels in the C-IKKα tumors, while those of Control and N-IKKα tumors are narrow and mature. Strong and delocalized suprabasal integrin-a6 staining is detected in N-IKKα tumors, while Control and C-IKKα tumors show Integrin-α6 basal expression. Reduced staining of Maspin in N-IKKα tumors. No difference in p52 and p65 expression was notice between tumors of the three genotypes.

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Staining, Marker, Control

Journal: Oncotarget

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

doi: 10.18632/oncotarget.8792

Figure Lengend Snippet: A-C. Immunofluorescence with a Flag specific antibody showing the expression of the transgene in the nucleus of the HaCaT-N-IKKα cells (B) and in the cytoplasm of the HaCaT-C-IKKα cells (C). D. Representative western blot analyisis showing increased levels of IKKα in different pools of transfected HaCaT clones. Observe the increased MMP-9 and EGFR activation in the HaCaT-C-IKKα cells and the enhanced expression of c-Myc in the HaCaT-N-IKKα cells. E. Graphic representation of the densitometric analysis of western blots correponding to 6 pooled clones of HaCaT-C-IKKα cells, 3 pooled clones of HaCaT-N-IKKα cells and 3 pooled clones of HaCaT-Control cells. Student's t test was used for statistical analysis. (*p<0.05; ****p<0.0001).

Article Snippet: The separated proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL; BioRad, France) and probed with antibodies against IKKα (NB100-56704 Novus Biologicals); c-Myc (Biolegend, CA, USA); Maspin, Actin, EGFR, P-EGFR (Tyr1176), p65, GAPDH (Santa Cruz Biotechnology, Inc. Europe); α-Tubulin (Sigma-Aldrich, MO, USA); E-Cadherin (BD Bioscience, NJ, USA). p100/p52 (Cell Signaling Technology, USA) and MMP-9 (Merck Millipore, Darmstadt, Germany).

Techniques: Immunofluorescence, Expressing, Western Blot, Transfection, Clone Assay, Activation Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells *

doi: 10.1074/jbc.M116.771014

Figure Lengend Snippet: Vorinostat-induced IL-8/CXCL8 expression is dependent on IKK activity and associated with IKKβ recruitment to the IL-8 promoter in ovarian cancer cells. A, Western blotting of CEs and NEs prepared from SKOV3 cells incubated with vorinostat for 48 h and analyzed by using p65, Lys-314/315 ac-p65, p50, IκBα, histone H3, Lys-9/14 ac-histone H3, and actin antibodies. Each lane corresponds to ∼5 × 104 cells. B, densitometric evaluation of IκBα levels in CEs and NEs (top panel) and of p65, Lys-314/315 ac-p65, p50, histone H3, and Lys-9/14 ac-histone H3 in NEs (bottom panel) of SKOV3 cells shown in A. The protein densities were normalized to actin. The values in untreated cells were arbitrarily set to 1, and the other values are presented relative to these values. The data represent the means of three experiments ± S.E. *, p < 0.05 compared with untreated cells. C and D, real-time RT-PCR analysis of IL-8 mRNA (C) and ELISA of the released IL-8 (D) in SKOV3 cells preincubated for 12 h with 5 μm Bay 117085, 5 mm aspirin, 10 μm SB 203580, or control DMSO and then treated for 48 h with 1.5 μm vorinostat. E, ChIP analysis of IKKα, IKKβ, and IKKϵ occupancy at the IL-8 promoter quantified by real-time PCR in SKOV3 cells incubated for 48 h with increasing concentrations of vorinostat. F, ChIP of IKKβ occupancy at the IL-8, TNFα, and IL-6 promoters in SKOV3 cells incubated for 48 h with vorinostat. The results in E and F are presented as -fold difference in occupancy over the human IGX1A (SA Biosciences) sequence control and represent the mean ± S.E. of three experiments. G, real-time RT-PCR analysis of IKKα, IKKβ, and IKKϵ mRNA levels in SKOV3 cells incubated for 48 h with increasing vorinostat. H, Western blotting analysis of IKKβ and control actin levels in WCEs of SKOV3 cells incubated for 48 h with increasing vorinostat (top panel). Each lane corresponds to ∼5 × 104 cells. Bottom panel, densitometric evaluation of IKKβ (shown in the top panel) normalized to actin. The values are presented relative to the value in untreated cells, which was set to 1. The data represent the means of three experiments ±S.E.

Article Snippet: The antibodies against human p65 NFκB (sc-372), p50 NFκB (sc-7178), IκBα (sc-371), histone H3 (sc-8654), Lys-9/14-acetylated histone H3 (sc-8655), IKKα (sc-7218), IKKβ (sc-8014), IKKε (sc-376114), IL-8/CXCL8 (sc-7922), CXCR1 (sc-7303), and CXCR2 (sc-7304) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Activity Assay, Western Blot, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control, Real-time Polymerase Chain Reaction, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells *

doi: 10.1074/jbc.M116.771014

Figure Lengend Snippet: Proposed model of IL-8 regulation by HDAC and IKK in ovarian cancer cells. In ovarian cancer cells, the IL-8 promoter is occupied by an HDAC, which limits IL-8 transcription. Inhibition of HDAC activity increases IKKβ recruitment to the IL-8 promoter, facilitating recruitment of p65 NFκB and its acetylation, resulting in increased IL-8 transcription, cell survival, and proliferation. Inhibition of IKK activity decreases p65 recruitment to the IL-8 promoter, suppressing HDI-induced IL-8 expression.

Article Snippet: The antibodies against human p65 NFκB (sc-372), p50 NFκB (sc-7178), IκBα (sc-371), histone H3 (sc-8654), Lys-9/14-acetylated histone H3 (sc-8655), IKKα (sc-7218), IKKβ (sc-8014), IKKε (sc-376114), IL-8/CXCL8 (sc-7922), CXCR1 (sc-7303), and CXCR2 (sc-7304) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Activity Assay, Expressing