Journal:

Article Title: Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori

doi:

Figure Lengend Snippet: (A) Detection of stable MKN45 transfectants expressing IκBα (SS32/36AA). The clone was analysed using a monoclonal antibody to FLAG and polyclonal antibody to IκBα. (B) Specific protein binding activities of nuclear factor kappa B (NFκB) sequences (electrophoretic mobility shift assay). The nuclear extracts were prepared from MKN45 and MKN45 IκBα SR cells. Cells were treated or not treated with Helicobacter pylori (HP) or 10 ng/ml tumour necrosis factor α (TNF-α) for 90 minutes. Nuclear extracts was incubated with 32P labelled oligonucleotide for 30 minutes. Migration of the DNA-protein complex containing NFκB is indicated. This complex was found to be specific, as judged using supershifting antibody against p65 and cold NFκB probes. (C) MKN45 and MKN45 IκBα SR cells were treated with interferon γ (IFN-γ 10 ng/ml) for 24 hours. Cells were incubated with H pylori (HP) or anti-Fas (CH-11) for 24 hours. Cell viability was assessed by trypan blue dye exclusion assay by counting 300 cells. Viable cells were quantitated under bright field microscopy. Results are expressed as percentages of dead cells. Values are mean (SD) of three independent experiments. *Percentage cytotoxicity was significantly (p<0.05) different between the MKN45 treated cells with anti-Fas or cocultured with H pylori and MKN45 IκBα SR treated cells with anti-Fas or cocultured with H pylori. (D) DNA fragmentation was quantified using a commercially available ELISA (Boehringer Mannheim Biochemicals, Mannheim, Germany): 5×104 cells were incubated in triplicate with H pylori (HP), anti-Fas (CH-11), or medium alone for 12 hours and lysed, and the supernatants were used for ELISA. Absorbance was measured at 405 nm. *Absorbance was significantly (p<0.05) different between the control and treated with anti-Fas or cocultured with H pylori in the MKN45 IκBα SR groups; NS, no significant difference was found. (E) MKN45 and MKN-45 IκBαSR cells were treated with IFN-γ (10 ng/ml) for 24 hours. Cells were incubated with H pylori (HP) or anti-Fas (CH-11) for 12 hours. Immunoblot analysis was performed using anti-caspase-8, caspase-3, cleaved caspase-3, and anti-actin antibody. (F) MKN45 and THP-1 cells were incubated with H pylori and total RNA was extracted at the indicated times. The ribonuclease protection assay was performed according to the supplier’s instructions.

Article Snippet: Detection of NFκB was performed with a 32 P dATP labelled oligo probe containing the NFκB recognition site purchased from Promega (Madison, Wisconsin, USA).

Techniques: Expressing, Protein Binding, Electrophoretic Mobility Shift Assay, Incubation, Migration, Exclusion Assay, Microscopy, Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: NF-κB p65 regulates NOX4 transcription via promoter binding and facilitates poly-PR 20 -induced NOX4 expression. (A–E) RT-qPCR analysis of NOX4 mRNA levels in astrocytes following overexpression of the transcription factors SOX2 (A), FOS (B), OCT1 (C), SP3 (D), and NF-κB p65 (E). Only p65 significantly enhanced NOX4 mRNA expression. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗∗∗ p < 0.001). N = 3 biologically independent experiments with consistent results. (F) Schematic diagram illustrating the experimental design for assessing NOX4 mRNA expression, protein levels, and luciferase reporter activity following poly-PR 20 treatment and p65 overexpression. (G–I) Co-treatment with poly-PR 20 and p65 overexpression further increased NOX4 mRNA expression (G) and protein levels (H, I) compared to poly-PR 20 treatment alone. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Tukey's multiple comparisons test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results. (J) Schematic representation of predicted NF-κB p65-responsive elements within the NOX4 promoter and design of luciferase reporter constructs (NOX4/FL-pGL3: −710 to +1; NOX4/PI-pGL3: −210 to +1). (K) Luciferase reporter assay showing that p65 enhances transcriptional activity through the NOX4 promoter. Cells were co-transfected with HA-p65 and the indicated NOX4 reporter constructs. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Over Expression, Two Tailed Test, Luciferase, Activity Assay, Construct, Reporter Assay, Transfection

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: Exogenous poly-PR 20 activates p65 phosphorylation and nuclear translocation in astrocytes via the IKK/IκB pathway. (A) Western blot analysis of phosphorylated p65 (p-p65) and total p65 in astrocytes treated with poly-PR 20 . p65 phosphorylation was notably increased at 3 h post-treatment. (B) Quantification of the p-p65/p65 ratio from three biologically independent experiments, all yielding consistent results. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗ p < 0.05). (C) Immunofluorescence staining showing nuclear translocation of p65 in astrocytes treated with FITC-poly-PR 20 for 3 h. (D) Confocal microscopy with Z-stack and line-scan analysis confirmed p65 nuclear localization. Red: p65; Green: FITC-poly-PR 20 ; Blue: DAPI. (E) Semi-quantitative analysis of nuclear versus cytosolic p65 using ImageJ (version 1.53t) from confocal images. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗∗∗ p < 0.001). Total number of cells analyzed: control group, n = 24; FITC-poly-PR 20 group, n = 24. N = 3 biologically independent experiments with consistent results. (F) Western blot analysis showing activation of IKKα/β phosphorylation following poly-PR 20 treatment. (G, H) Quantification of phosphorylated IKKα/β relative to total IKKα (G) and IKKβ (H) revealed a significant increase at 30 min post-treatment. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results. (I) Western blot analysis showing increased phosphorylation of IκBα in astrocytes following poly-PR 20 treatment. (J) Quantification of the p -IκBα/IκBα ratio showed a significant increase at 2 h. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). N = 3 biologically independent experiments with consistent results.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Two Tailed Test, Control, Activation Assay

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: Sigma-1R interacts with p65 and reduces its binding to the NOX4 promoter in astrocytes under exogenous poly-PR 20 peptide treatment. (A) Western blot analysis of phosphorylated p65 (p-p65) levels in astrocytes overexpressing Sigma-1R-GFP or GFP control, followed by exogenous poly-PR 20 peptide treatment. Astrocytes were transfected for 24 h and treated with exogenous poly-PR 20 peptide for 3 h. (B) Quantification of the p-p65/p65 protein ratio from three biologically independent experiments, each yielding consistent results. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test. (C) Schematic diagram illustrating the experimental timeline for the luciferase reporter assay following co-transfection of GFP or Sigma-1R-GFP with HA-p65, followed by exogenous poly-PR 20 peptide treatment. (D) Overexpression of Sigma-1R-GFP reduced p65-mediated activation of the NOX4 promoter. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗ p < 0.05). N = 3 biologically independent experiments with consistent results. (E) Schematic diagram illustrating the experimental timeline for the luciferase reporter assay after clemastine and poly-PR 20 treatment. (F) Clemastine treatment also reduced p65-driven NOX4 promoter activation. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗ p < 0.05). N = 3 biologically independent experiments with consistent results. (G) Immunoprecipitation analysis of p65 and Sigma-1R interaction in astrocytes treated with poly-PR 20 confirmed a physical interaction between Sigma-1R and p65. (H) Immunofluorescence analysis demonstrated that Sigma-1R overexpression in astrocytes attenuated p65 nuclear translocation in response to exogenous poly-PR peptide treatment. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t -test (∗∗∗∗ p < 0.0001). Total number of cells analyzed: EGFP + poly-PR 20 group, n = 18; Sigma-1R-EGFP + poly-PR 20 group, n = 13. N = 3 biologically independent experiments with consistent results.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Binding Assay, Western Blot, Control, Transfection, Two Tailed Test, Luciferase, Reporter Assay, Cotransfection, Over Expression, Activation Assay, Immunoprecipitation, Immunofluorescence, Translocation Assay

Journal: Redox Biology

Article Title: Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

doi: 10.1016/j.redox.2025.103875

Figure Lengend Snippet: Proposed model of Sigma-1R-mediated protection against exogenous poly-PR-induced oxidative stress in astrocytes. Neuron-derived poly-PR is released and internalized by astrocytes, where it activates the IKK/IκB/NF-κB pathway, driving p65 nuclear translocation, NOX4 transcription, and H 2 O 2 production. Sigma-1R overexpression interacts with p65, limiting its nuclear translocation and binding to the NOX4 promoter, thereby suppressing astrocytic oxidative stress and neuroinflammation. This model underscores the non-cell-autonomous neuron-to-astrocyte mechanism contributing to C9orf72 ALS pathogenesis.

Article Snippet: NFκB (p65) , Merck , 06-418; RRID: AB_11214166.

Techniques: Derivative Assay, Translocation Assay, Over Expression, Binding Assay