Journal:

Article Title: The NEMO Mutation Creating the Most-Upstream Premature Stop Codon Is Hypomorphic Because of a Reinitiation of Translation

doi:

Figure Lengend Snippet: NF-κB signaling in SV40-transformed fibroblasts. A, Time-course analysis of IκBα protein degradation and resynthesis detected by western blot after stimulation with IL-1β for 20, 40, 60, and 120 min; the antibody against p38 served as a control for protein loading. B, NF-κB DNA-binding activity, measured by EMSA after stimulation for 20 and 40 min with IL-1β. γ-Activating–factor DNA-binding activity after 20 min of stimulation with IFN-γ and competition with unlabeled NF-κB–specific probe (*) served as internal activation control and specificity control, respectively. C, IL-6 production after 18 h of exposure to IL-1β, TNF-α, and PMA/ionomycin, in fibroblasts from a healthy control, from the patient, from a patient with XR-OL-EDA-ID (X420W), and from a patient with XD-IP (ex4_10del). Results shown are representative of two to three independent experiments.

Article Snippet: The rabbit anti-human IKKα (H-744, sc-7218), mouse anti-human IKKβ (H-4, sc-8014), mouse anti-human STAT-2 (A-7, sc-1668), rabbit anti-mouse p38 (C-20, sc-535), mouse anti-human IKKγ (B-3, sc-8032), goat anti-human IKKγ (M-18, sc-8256), and rabbit anti-human IκBα (C-21, sc-37) antibodies were purchased from SantaCruz Biotechnology (Tebu-Bio S.A.).

Techniques: Transformation Assay, Western Blot, Control, Binding Assay, Activity Assay, Activation Assay

Journal:

Article Title: The NEMO Mutation Creating the Most-Upstream Premature Stop Codon Is Hypomorphic Because of a Reinitiation of Translation

doi:

Figure Lengend Snippet: NEMO production in SV40-transformed fibroblasts. A, NEMO production assessed by western blot analysis, with the use of antibodies directed against the N- or C-terminal part of NEMO (amino acids 28–33 and 356–414, respectively), antibodies against p38 and STAT-2 being used as controls for protein loading. B, Intracellular staining with an antibody directed against amino acids 278–396 of NEMO (black) or an isotype-control antibody (gray) of fibroblasts from a control, from the patient, from a patient with XR-OL-EDA-ID, and from a patient with XD-IP. Results shown are representative of three independent experiments.

Article Snippet: The rabbit anti-human IKKα (H-744, sc-7218), mouse anti-human IKKβ (H-4, sc-8014), mouse anti-human STAT-2 (A-7, sc-1668), rabbit anti-mouse p38 (C-20, sc-535), mouse anti-human IKKγ (B-3, sc-8032), goat anti-human IKKγ (M-18, sc-8256), and rabbit anti-human IκBα (C-21, sc-37) antibodies were purchased from SantaCruz Biotechnology (Tebu-Bio S.A.).

Techniques: Transformation Assay, Western Blot, Staining, Control

Journal:

Article Title: The NEMO Mutation Creating the Most-Upstream Premature Stop Codon Is Hypomorphic Because of a Reinitiation of Translation

doi:

Figure Lengend Snippet: Expression of various NEMO constructs in vitro. A, WT sequence of the 5′ end of NEMO showing the first methionine site involved in the initiation of translation (M1, boxed), the position of the 110_111insC mutation, the second methionine site (M38) involved in translation reinitiation, and the IP nonsense mutation at Arg 62 (R62X). B, Western blot analysis with an anti-V5 antibody of 293T HEK cells transfected with insert-free V5-tagged vector (mock) or with expression vectors carrying various NEMO alleles: WT, 110_111insC (the patient’s mutation), M38A (the methionine 38 to alanine mutation, blocking reinitiation of translation at M38) with or without the 110_111insC mutation, X420W, and R62X. Cells were cotransfected with 3 μg of each vector and 1 μg of a myc-tagged expression vector encoding β-galactosidase, as a transfection-efficiency control. An antibody against p38 was used to control for protein loading.

Article Snippet: The rabbit anti-human IKKα (H-744, sc-7218), mouse anti-human IKKβ (H-4, sc-8014), mouse anti-human STAT-2 (A-7, sc-1668), rabbit anti-mouse p38 (C-20, sc-535), mouse anti-human IKKγ (B-3, sc-8032), goat anti-human IKKγ (M-18, sc-8256), and rabbit anti-human IκBα (C-21, sc-37) antibodies were purchased from SantaCruz Biotechnology (Tebu-Bio S.A.).

Techniques: Expressing, Construct, In Vitro, Sequencing, Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Blocking Assay, Control

Journal: Cancer Research

Article Title: c-Jun NH2-Terminal Kinase–Related Na+/H+ Exchanger Isoform 1 Activation Controls Hexokinase II Expression in Benzo(a)Pyrene-Induced Apoptosis

doi: 10.1158/0008-5472.can-06-2327

Figure Lengend Snippet: Figure 2. MKK4/JNK pathway is involved in NHE1-related alkalinization in contrast to p53. A, F258 cells were treated or not with 50 nmol/L BaP and/or JNKi (1 Amol/L) or PFT (5 Amol/L) for 48 h. Resting pHi measurements were monitored by microspectrofluorimetry using the pH- sensitive fluorescent probe carboxy-SNARF-1 in HEPES- buffered medium. pHi values were derived from a pH calibration curve (as described in Materials and Methods) and DpHi was calculated by subtracting pHi values in treated cells from those in untreated cells. *, P < 0.05. Number in brackets, number of coverslips used (i.e., number of measurements done) from at least three independent experiments. Mean values correspond to the average of all pHi determinations. Basal pHi was 7.45 F 0.09 (n = 12), 7.45 F 0.05 (n = 12), and 7.48 F 0.08 (n = 7) in control untreated, JNKi-treated, or PFT-treated cells, respectively. Subsequently, pHi recovery rate was monitored following an acid load induced on removal of NH4Cl (20 mmol/L) from extracellular medium (see Materials and Methods). Recordings were obtained from BaP-treated cells versus control (top) and BaP + JNKi– treated cells versus JNKi control (bottom). B and C, cells were transfected with Si Neg or Si MKK4 (B) or Si p53 (C) and subsequently treated with 5 Amol/L BaP during 12 h. Resting pHi measurements were monitored and DpHi was calculated by subtracting pHi values in BaP-treated, Si MKK4–transfected, Si p53–transfected, or Si Neg–transfected cells from those in untreated, Si MKK4–transfected, Si p53–transfected, or Si Neg–transfected cells. *, P < 0.05. Number in brackets, number of coverslips used (i.e., number of measurements done) from at least three independent experiments. Mean values correspond to the average of all pHi determinations. C, 60 Ag of whole-cell lysate were separated on 10% SDS-PAGE. Immunoblot was probed with mouse monoclonal anti-p53, HSC70, and rabbit polyclonal anti–phosphorylated p53 (Ser15; P-p53) antibodies. Data are representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti–phosphorylated p53 (Ser15), anti-HKII, anti-MKP1, mouse monoclonal anti-h-actin, and anti-HSC70 antibodies were from Santa Cruz Biotechnology (Tebu-bio SA, Le Perray en Yvelynes, France).

Techniques: Derivative Assay, Control, Transfection, SDS Page, Western Blot

Journal: Cancer Research

Article Title: c-Jun NH2-Terminal Kinase–Related Na+/H+ Exchanger Isoform 1 Activation Controls Hexokinase II Expression in Benzo(a)Pyrene-Induced Apoptosis

doi: 10.1158/0008-5472.can-06-2327

Figure Lengend Snippet: Figure 3. NHE1 does not trigger p53 activation but is involved in superoxide anion production. A, F258 cells were transfected with Si Neg or Si NHE1. Si NHE1 efficiency was tested by expression analysis by RT-PCR after 48 h of transfection using 18S gene expression as control or by functional test by pHi recovery analysis following an acid load (see Materials and Methods). Recordings were obtained from Si NHE1–transfected versus Si Neg–transfected cells. Apoptotic nuclei (analyzed by Hoechst 33342 staining) and DEVDase activity measurements were done following a 48-h treatment with 5 Amol/L BaP in Si Neg–transfected, Si p53– transfected, and Si NHE1–transfected cells (n = 3 independent experiments). B, F258 cells were treated with 50 nmol/L or 5 Amol/L of BaP with or without 30 Amol/L cariporide (Caripo), or cells were transfected with Si Neg or Si NHE1 and treated with 5 Amol/L BaP during 48 h. Whole-cell lysate (60 Ag) was separated on 12.5% SDS-PAGE. Immunoblot was probed with a rabbit polyclonal anti– phosphorylated p53 (Ser15) antibody. Data are representative of four independent experiments. After fixation and permeabilization, immunostaining of cells was done with rabbit polyclonal anti– phosphorylated p53 (Ser15) antibody and with an antirabbit antibody coupled to TRITC (red). Cells were costained with DAPI (blue) to detect nuclei and subsequently viewed by fluorescence microscopy. Magnification, 400. The experiments were repeated at least thrice with similar results. Bar, 4 Am. C, production of ROS was assessed by flow cytometry analysis. F258 cells were treated (black line) or not (gray peak) with 50 nmol/L BaP in the presence or absence of JNKi (1 Amol/L), NHE1 inhibitor (30 Amol/L cariporide), and p53 translocation inhibitor (5 Amol/L PFT) during 48 h, or F258 cells were transfected with Si Neg, Si NHE1, or Si p53 and treated with 500 nmol/L BaP during 48 h. O2 was detected with DHE probe. Menadione (100 Amol/L) was used as positive control. The experiments were repeated at least thrice with similar results.

Article Snippet: Rabbit polyclonal anti–phosphorylated p53 (Ser15), anti-HKII, anti-MKP1, mouse monoclonal anti-h-actin, and anti-HSC70 antibodies were from Santa Cruz Biotechnology (Tebu-bio SA, Le Perray en Yvelynes, France).

Techniques: Activation Assay, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control, Functional Assay, Staining, Activity Assay, SDS Page, Western Blot, Immunostaining, Fluorescence, Microscopy, Flow Cytometry, Translocation Assay, Positive Control

Journal: Cancer Research

Article Title: c-Jun NH2-Terminal Kinase–Related Na+/H+ Exchanger Isoform 1 Activation Controls Hexokinase II Expression in Benzo(a)Pyrene-Induced Apoptosis

doi: 10.1158/0008-5472.can-06-2327

Figure Lengend Snippet: Figure 5. BaP-induced NHE1 activation affects mitochondrial HKII expression, which promotes apoptosis. A, cells were first transfected with Si Neg or Si HKII B66 or Si HKII B67 and subsequently treated with 5 Amol/L BaP during 48 h. A Western blot analysis of HKII was done on control total cell lysates following 48 h after transfection to estimate the efficacy of silencing. Apoptotic nuclei analyzed by Hoechst 33342 staining. *, P < 0.05, BaP-treated cells versus control cells; #, P < 0.05, BaP-treated Si HKII–transfected cells versus BaP-treated Si Neg–transfected cells (n = 3 independent experiments). Quantitative analysis of HKII protein expression by densitometry is given in the lowest histogram. The results are expressed as the ratio of HKII to that of total h-actin. Data are representative of three independent experiments. *, P < 0.05, Si HKII–transfected versus Si Neg–transfected cells. B, F258 cells were transfected or not with Si Neg, Si p53, or Si NHE1 and treated or not with 5 Amol/L BaP during 48 h. After fixation and permeabilization, immunostaining of cells was done with a rabbit polyclonal anti-HKII antibody by using as secondary antibody an anti-rabbit coupled to FITC (green) and with MitoTracker Red CMXROS. Cells were costained with DAPI (blue) to detect nuclei and subsequently viewed by fluorescence microscopy. Magnification, 400. The experiments were repeated at least thrice with similar results. Yellow, colocalization. Bar, 4 Am. C, Western blot analysis of HKII. F258 cells were treated with 50 nmol/L BaP alone or in the presence of a-NF (10 Amol/L), PFT (5 Amol/L), cariporide (30 Amol/L), or 5ThioG (5 mmol/L) during 48 h, or cells were transfected Si Neg, Si p53, or Si NHE1 and treated or not with 5 Amol/L BaP during 48 h. Mitochondrial fractions (50 Ag; see Materials and Methods) were separated on 12% SDS-PAGE. Immunoblot was probed with rabbit polyclonal anti-HKII antibody and with mouse monoclonal anti-COX IV antibody as a loading control. Data are representative of three independent experiments. D, analysis of HKII expression was done by RT-PCR following a 48-h treatment with or without BaP (50 nmol/L) in the presence or absence of cariporide (30 Amol/L). Top, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used as a control insensitive to BaP. F258 cells were transfected with Si Neg, Si p53, or Si NHE1, and RT-PCR was done after treatment with 5 Amol/L BaP during 48 h. Bottom, 18S gene expression was used as a control.

Article Snippet: Rabbit polyclonal anti–phosphorylated p53 (Ser15), anti-HKII, anti-MKP1, mouse monoclonal anti-h-actin, and anti-HSC70 antibodies were from Santa Cruz Biotechnology (Tebu-bio SA, Le Perray en Yvelynes, France).

Techniques: Activation Assay, Expressing, Transfection, Western Blot, Control, Staining, Immunostaining, Fluorescence, Microscopy, SDS Page, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Cancer Research

Article Title: c-Jun NH2-Terminal Kinase–Related Na+/H+ Exchanger Isoform 1 Activation Controls Hexokinase II Expression in Benzo(a)Pyrene-Induced Apoptosis

doi: 10.1158/0008-5472.can-06-2327

Figure Lengend Snippet: Figure 6. Schematic summary of the origin and role of NHE1 activation in BaP-induced apoptosis. BaP, a lipophilic molecule, goes across the bilayer membrane. In the cytosol, BaP is metabolized by CYP1 into reactive metabolites, which form DNA adducts, leading to p53 activation. The CYP1-dependent metabolism leads to an early production of H2O2, which activates NHE1 via MKK4/ JNK recruitment. Both NHE1 and p53 pathways would target HKII present in mitochondria, leading to the production of O2 by the complex III. These mitochondrial dysfunctions would promote late apoptotic events, such as acidification and caspase activation. Caspase-independent processes, shown to be triggered by p53, would also occur depending or not on acidification (11).

Article Snippet: Rabbit polyclonal anti–phosphorylated p53 (Ser15), anti-HKII, anti-MKP1, mouse monoclonal anti-h-actin, and anti-HSC70 antibodies were from Santa Cruz Biotechnology (Tebu-bio SA, Le Perray en Yvelynes, France).

Techniques: Activation Assay, Membrane