Journal: microLife

Article Title: Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ

doi: 10.1093/femsml/uqaf049

Figure Lengend Snippet: Cortactin gene knockout results in reduced activation of NF-κB associated with reduced phospho-NF-κB p65 (S-536) recruitment into AGS cell nuclei upon H. pylori infection. Time course of NF-κB activation over 24 h in (A) AGS wt and (B) AGSΔ cttn knockout cells during infection with the indicated H. pylori strains. Quantification of NF-κB activity by SEAP reporter assay (Quanti Blue) reveals enhanced NF-κB activity in wt vs. Δ cttn AGS cells, which depends on the functional cag type IV secretion system and the effector molecules ADPH and CagA. (C) AGS cells were incubated without (Mock) or with H. pylori for 4 h, and then stained for phospho-NF-κB p65 (S-536) (green) and nuclei (blue). As revealed by fluorescence microscopy, H. pylori provoke strong activation of NF-κB p65 in wt (red arrowheads), but not in Δ cttn cells. (D) Representative AGS wt and Δ cttn cells from panel A (cells in white boxes) showing phospho-NF-κB p65 accumulation (green) within the nuclei upon infection with wt H. pylori . Nuclear and cellular margins are indicated by red and white dashed lines, respectively. (E) Relative fluorescence intensity (RFU) of nuclear phospho-NF-κB p65 was quantified. Mean values ± standard error are shown with significant difference *** corresponding to P ≤ .001.

Article Snippet: Other antibodies used in this work were specific for detecting CagY (Tegtmeyer et al. ), UreB (Tegtmeyer et al. ), cortactin (#05–180, Merck-Millipore), Src (#2123, Cell Signaling Technology, Frankfurt, Germany), GAPDH (#sc-47724, Santa Cruz Biotechnology), TIFA (#61358, Cell Signaling Technology), phospho-TIFA-T9 (#ab214815, Abcam, Cambridge, UK), IKKβ (#05–535, clone 10AG2, Merck-Millipore), phospho-IKKβ-Y199 (#59195, Abcam), phospho-NF-κB p65-S536 (#3031, Cell Signaling Technology), FAK (#610087, BD Bioscience, Heidelberg, Germany), phospho-FAK-Y397 (#3283S, Cell Signaling Technology), and phospho-Src-Y418 (#OP07, Oncogene, Heidelberg, Germany). α-rabbit (#31462) or α-mouse (#31446) polyvalent goat antibodies conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (Thermo Fisher Scientific).

Techniques: Gene Knockout, Activation Assay, Infection, Knock-Out, Activity Assay, Reporter Assay, Functional Assay, Incubation, Staining, Fluorescence, Microscopy

Journal: microLife

Article Title: Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ

doi: 10.1093/femsml/uqaf049

Figure Lengend Snippet: AGS cells with cortactin deficiency demonstrate diminished NF-κB p65 and IL-8 cellular levels in response to H. pylori infection. (A) AGS wt and (B) AGSΔ cttn knockout cells were transfected with pNF-κB p65-YFP (yellow) and pIL-8-GFP (green) constructs for 24 h followed by 4 h incubation without (mock) or with wt H. pylori . After fixation, cells were stained with DAPI and phalloidin to visualize cell nuclei (blue) and filamentous actin (gray), respectively. White arrows indicate cellular expression of NF-κB p65-YFP in the absence of H. pylori , while red arrows show increased levels of NF-κB p65-YFP and pIL-8-GFP in the presence of the pathogen. (C) RFU of pIL-8-GFP in transfected AGS wt vs. Δ cttn cells were quantified. The values in AGS wt cells were set to 100%. Mean values ± standard error are shown with significant difference * corresponding to P ≤ .05.

Article Snippet: Other antibodies used in this work were specific for detecting CagY (Tegtmeyer et al. ), UreB (Tegtmeyer et al. ), cortactin (#05–180, Merck-Millipore), Src (#2123, Cell Signaling Technology, Frankfurt, Germany), GAPDH (#sc-47724, Santa Cruz Biotechnology), TIFA (#61358, Cell Signaling Technology), phospho-TIFA-T9 (#ab214815, Abcam, Cambridge, UK), IKKβ (#05–535, clone 10AG2, Merck-Millipore), phospho-IKKβ-Y199 (#59195, Abcam), phospho-NF-κB p65-S536 (#3031, Cell Signaling Technology), FAK (#610087, BD Bioscience, Heidelberg, Germany), phospho-FAK-Y397 (#3283S, Cell Signaling Technology), and phospho-Src-Y418 (#OP07, Oncogene, Heidelberg, Germany). α-rabbit (#31462) or α-mouse (#31446) polyvalent goat antibodies conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (Thermo Fisher Scientific).

Techniques: Infection, Knock-Out, Transfection, Construct, Incubation, Staining, Expressing

Journal: microLife

Article Title: Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ

doi: 10.1093/femsml/uqaf049

Figure Lengend Snippet: TIFA and NF-κB signaling activation in AGS cells upon H. pylori infection. (A) AGS wt or AGSΔ cttn knockout cells were infected with the indicated H. pylori strains for 45 min. Protein lysates were subjected to IP with anti-TIFA antibodies. IP samples against total TIFA show equal amounts of TIFA present across all samples. Probing for phospho-TIFA (Threonine 9) demonstrates activation of TIFA in H. pylori wt and Δ cagA mutant-infected samples (lanes 2 and 5), while no activation is observed in mock, Δ cag PAI, Δ cagY , or Δg mhA infections (lanes 1, 3, 4, and 6). (B) Quantification of TIFA phospho-band intensities from panel A. (C) Total protein lysates prepared from infected cells after 45 min were subjected to Western blotting against bacterial proteins (Urease B and CagA) as well as host cell total cortactin, IKKβ and GAPDH, which confirms similar expression levels across all samples. Phospho-specific blots against IKKβ (Y-199) and p65 (S-536) revealed their pronounced activation only in lanes 2 and 5, as expected. Quantification of phospho-IKKβ (D) and phospho-p65 (E) band intensities of panel C. The data are presented as relative band intensity values. (F) Quantification of NF-κB activity by SEAP reporter assay (Quanti Blue) and (G) IL-8 secretion by standard ELISA in the same set of experiments. These studies revealed increased NF-κB activity and IL-8 production in cells infected for 6 h with H. pylori wt and Δ cagA mutant strains. Mean values ± standard error are shown with significant difference *** corresponding to P ≤ .001 and * to ≤ .05.

Article Snippet: Other antibodies used in this work were specific for detecting CagY (Tegtmeyer et al. ), UreB (Tegtmeyer et al. ), cortactin (#05–180, Merck-Millipore), Src (#2123, Cell Signaling Technology, Frankfurt, Germany), GAPDH (#sc-47724, Santa Cruz Biotechnology), TIFA (#61358, Cell Signaling Technology), phospho-TIFA-T9 (#ab214815, Abcam, Cambridge, UK), IKKβ (#05–535, clone 10AG2, Merck-Millipore), phospho-IKKβ-Y199 (#59195, Abcam), phospho-NF-κB p65-S536 (#3031, Cell Signaling Technology), FAK (#610087, BD Bioscience, Heidelberg, Germany), phospho-FAK-Y397 (#3283S, Cell Signaling Technology), and phospho-Src-Y418 (#OP07, Oncogene, Heidelberg, Germany). α-rabbit (#31462) or α-mouse (#31446) polyvalent goat antibodies conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (Thermo Fisher Scientific).

Techniques: Activation Assay, Infection, Knock-Out, Mutagenesis, Western Blot, Expressing, Activity Assay, Reporter Assay, Enzyme-linked Immunosorbent Assay

Journal: microLife

Article Title: Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ

doi: 10.1093/femsml/uqaf049

Figure Lengend Snippet: Effects of ADPH and CagA on phosphorylation of FAK, Src, IKKβ, and p65 NF-κB in AGS wt vs. cortactin knockout cells. The indicated cells were transfected with CagA and/or treated with synthetic ADPH as described in the section “Materials and methods.” (A) Protein lysates were subjected to Western blotting and probed with the indicated antibodies against CagA, total FAK, and total Src as loading controls. (B) Protein lysates from panel A were probed with the indicated phospho-specific antibodies. The blots show that phospho-FAK (Y-397) and phospho-Src (Y-418) bands are CagA-dependent and absent in Δ cttn cells. Activation of phospho-IKKβ (Y-199) and phospho-p65 (S-536) is strong upon CagA expression and further enhanced by combined CagA transfection and ADPH treatment. (C–F) Quantification of phospho-FAK, phospho-Src, phospho-IKKβ, and phospho-p65 band intensities shown in panel B. Mean values ± standard error are shown with significant difference *** corresponding to P ≤ .001.

Article Snippet: Other antibodies used in this work were specific for detecting CagY (Tegtmeyer et al. ), UreB (Tegtmeyer et al. ), cortactin (#05–180, Merck-Millipore), Src (#2123, Cell Signaling Technology, Frankfurt, Germany), GAPDH (#sc-47724, Santa Cruz Biotechnology), TIFA (#61358, Cell Signaling Technology), phospho-TIFA-T9 (#ab214815, Abcam, Cambridge, UK), IKKβ (#05–535, clone 10AG2, Merck-Millipore), phospho-IKKβ-Y199 (#59195, Abcam), phospho-NF-κB p65-S536 (#3031, Cell Signaling Technology), FAK (#610087, BD Bioscience, Heidelberg, Germany), phospho-FAK-Y397 (#3283S, Cell Signaling Technology), and phospho-Src-Y418 (#OP07, Oncogene, Heidelberg, Germany). α-rabbit (#31462) or α-mouse (#31446) polyvalent goat antibodies conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (Thermo Fisher Scientific).

Techniques: Phospho-proteomics, Knock-Out, Transfection, Western Blot, Activation Assay, Expressing

Journal: microLife

Article Title: Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ

doi: 10.1093/femsml/uqaf049

Figure Lengend Snippet: NF-κB activation in AGS wt and knockout cells for cortactin, FAK and TIFA by transfection of CagA and addition of ADPH. (A) AGS wt cells and knockout cells of (B) cortactin, (C) FAK, or (D) TIFA were transfected with CagA and/or incubated with ADPH as described in the section “Materials and methods”. Western blotting against CagA, cortactin, FAK, and TIFA confirmed similar protein expression in each sample or their corresponding absence. (E–H) Quantification of NF-κB activation by Quanti-Blue assay revealed cummulative effects of CagA and ADPH in wt cells (lane 4). Treatment of cortactin or FAK knockout cells resulted in a similar NF-κB activation by ADPH, but not CagA, while in TIFA knockout cells ADPH was unable to induce NF-κB activation showing only CagA-dependent effects. (I–L) These data correlated with the production of IL-8 as determined by standard ELISA in the same experiments. Mean values ± standard error are shown with significant difference *** corresponding to P ≤ .001; n.s. not significant.

Article Snippet: Other antibodies used in this work were specific for detecting CagY (Tegtmeyer et al. ), UreB (Tegtmeyer et al. ), cortactin (#05–180, Merck-Millipore), Src (#2123, Cell Signaling Technology, Frankfurt, Germany), GAPDH (#sc-47724, Santa Cruz Biotechnology), TIFA (#61358, Cell Signaling Technology), phospho-TIFA-T9 (#ab214815, Abcam, Cambridge, UK), IKKβ (#05–535, clone 10AG2, Merck-Millipore), phospho-IKKβ-Y199 (#59195, Abcam), phospho-NF-κB p65-S536 (#3031, Cell Signaling Technology), FAK (#610087, BD Bioscience, Heidelberg, Germany), phospho-FAK-Y397 (#3283S, Cell Signaling Technology), and phospho-Src-Y418 (#OP07, Oncogene, Heidelberg, Germany). α-rabbit (#31462) or α-mouse (#31446) polyvalent goat antibodies conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (Thermo Fisher Scientific).

Techniques: Activation Assay, Knock-Out, Transfection, Incubation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: microLife

Article Title: Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ

doi: 10.1093/femsml/uqaf049

Figure Lengend Snippet: Expression of CagA promotes NF-κB activation in MEFs in a cortactin-, ERK1/2 kinase-, and Src-dependent manner. Primary MEF cells from (A) wt or (B–D) cortactin knockout mice were transfected with CagA and treated with PD98051 or PP2, pharmacological inhibitors of ERK1/2 and Src kinases, respectively. (C and D) Cortactin-deficient MEFs were additionally transfected with GFP-cortactin expression plasmids (wt or phosphorylation-deficient mutant S405/418A). (A–D) Western blotting against CagA, cortactin, GFP, and GAPDH showed similar expression of CagA and cortactin proteins in the samples. The arrow marks the GFP-cortactin fusion proteins, and the arrowhead marks endogenous cortactin proteins. (E–H) Quantification of NF-κB activity by Quanti-Blue assay after 12 h. (I–L) Quantification of KC secretion by ELISA of the respective samples shows correlation with NF-κB activation. Mean values ± standard error are shown with significant difference *** corresponding to P ≤ .001; n.s. not significant.

Article Snippet: Other antibodies used in this work were specific for detecting CagY (Tegtmeyer et al. ), UreB (Tegtmeyer et al. ), cortactin (#05–180, Merck-Millipore), Src (#2123, Cell Signaling Technology, Frankfurt, Germany), GAPDH (#sc-47724, Santa Cruz Biotechnology), TIFA (#61358, Cell Signaling Technology), phospho-TIFA-T9 (#ab214815, Abcam, Cambridge, UK), IKKβ (#05–535, clone 10AG2, Merck-Millipore), phospho-IKKβ-Y199 (#59195, Abcam), phospho-NF-κB p65-S536 (#3031, Cell Signaling Technology), FAK (#610087, BD Bioscience, Heidelberg, Germany), phospho-FAK-Y397 (#3283S, Cell Signaling Technology), and phospho-Src-Y418 (#OP07, Oncogene, Heidelberg, Germany). α-rabbit (#31462) or α-mouse (#31446) polyvalent goat antibodies conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Activation Assay, Knock-Out, Transfection, Phospho-proteomics, Mutagenesis, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: microLife

Article Title: Enhanced ADP-heptose-dependent NF-κB activation by Helicobacter pylori CagA through cortactin-Src-dependent tyrosine phosphorylation of IKKβ

doi: 10.1093/femsml/uqaf049

Figure Lengend Snippet: Model of NF-κB signaling induced by H. pylori in (A) wt or (B) Δ cttn knockout cells. The H. pylori T4SS can inject two major substrates, CagA and ADPH, with the latter being responsible for the main portion of proinflammatory signaling through transcription factor NF-κB. In this scenario, CagA appears as a “helping molecule” by enhancing the ADPH response. In particular, translocated ADPH binds to a novel intracellular pathogen recognition receptor (PRR), cytoplasmic alpha kinase-1 (ALPK1). ALPK1 becomes autophosphorylated and then phosphorylates the adapter protein TIFA. TIFA then oligomerizes and forms the so-called TIFAsome. The activated TIFAsome in turn activates TAK kinase and the IKK complex. This IKK complex controls the downstream NF-κB complex, and p65 further translocates into the nucleus and activates IL-8 gene transcription. We hypothesize that injected CagA exhibits a supporting function in this signaling by stimulating ERK1/2-mediated serine phosphorylation of cortactin and tyrosine phosphorylation of FAK (Tegtmeyer et al. ). FAK and Src can interact and further activate each other. Active Src can then phosphorylate IKKβ (Y-199), which fully activates the IKK complex and overall NF-κB activity. When cortactin is absent, as in AGS or MEF Δ cttn knockout cells, NF-κB activity is downregulated by the portion contributed by CagA>cortactin>FAK>Src>IKKβ-mediated signaling as indicated in panel B.

Article Snippet: Other antibodies used in this work were specific for detecting CagY (Tegtmeyer et al. ), UreB (Tegtmeyer et al. ), cortactin (#05–180, Merck-Millipore), Src (#2123, Cell Signaling Technology, Frankfurt, Germany), GAPDH (#sc-47724, Santa Cruz Biotechnology), TIFA (#61358, Cell Signaling Technology), phospho-TIFA-T9 (#ab214815, Abcam, Cambridge, UK), IKKβ (#05–535, clone 10AG2, Merck-Millipore), phospho-IKKβ-Y199 (#59195, Abcam), phospho-NF-κB p65-S536 (#3031, Cell Signaling Technology), FAK (#610087, BD Bioscience, Heidelberg, Germany), phospho-FAK-Y397 (#3283S, Cell Signaling Technology), and phospho-Src-Y418 (#OP07, Oncogene, Heidelberg, Germany). α-rabbit (#31462) or α-mouse (#31446) polyvalent goat antibodies conjugated with horseradish peroxidase (HRP) were used as secondary antibodies (Thermo Fisher Scientific).

Techniques: Knock-Out, Injection, Phospho-proteomics, Activity Assay

Journal: Scientific Reports

Article Title: Loss of p190A RhoGAP induces aneuploidy and enhances bladder cancer cell migration and invasion by modulating actin dynamics

doi: 10.1038/s41598-025-23687-4

Figure Lengend Snippet: A p190A regulates LIMK/Cofilin and FAK/cortactin pathways in BC cells. (A) Western blots show that the expression of RhoA, ROCK1, phospho-LIMK1/2 (p-LIMK1/2), and phospho-cofilin1 (p-cofilin1) are upregulated upon p190A knockdown in RT4 cells using three different shRNAs (shp190A-1, shp190A-2, shp190A-3), while total cofilin and LIMK levels remained unchanged. Similarly, phospho-cortactin (p-cortactin) and phospho-FAK (p-FAK) levels are increased in cells with p190A knockdown but the total cortactin and FAK levels remain unchanged. (B) Western blots show that the expression of RhoA, ROCK1, phospho-LIMK1/2 (p-LIMK1/2), and phospho-cofilin1 (p-cofilin1) are downregulated upon ectopic p190A expression in BFTC cells (p190A-OE), while p190A overexpression did not influence total cofilin and LIMK levels. Similarly, phospho-cortactin (p-cortactin) and phospho-FAK (p-FAK) levels are downregulated upon p190A overexpression (BFTC-OE) but the total cortactin and FAK levels remain unchanged. α-tubulin, β-actin and GAPDH were used as loading controls. Please note that for all Western blots (WB), full-length gels are provided in the supplemental data file (Suppl. Figure ). Quantification of all WBs shown in Fig. 5, including full gels of the WBs are provided as supplemental data (Suppl. Figure ). The Western blots in Figs. , and 5 were repeated at least for three times (Suppl. Figure ). Please note that due to the experimental requirements, i.e. multiple use of the membranes with different antibodies, different loading controls were used in some cases in Figs. , and 5.

Article Snippet: For cortactin staining, RT4, T24, BFTC cells were incubated with mouse anti-Cortactin primary antibody (1:500, sc-55579, Santa Cruz, California, USA) overnight at 4°C.

Techniques: Western Blot, Expressing, Knockdown, Over Expression

Journal: Scientific Reports

Article Title: Loss of p190A RhoGAP induces aneuploidy and enhances bladder cancer cell migration and invasion by modulating actin dynamics

doi: 10.1038/s41598-025-23687-4

Figure Lengend Snippet: A p190A reduces invadopodia formation and gelatin degradation in BC cells. (A) Representative immunofluorescence (IF) images and quantification of invadopodia formation in bladder cancer cells. Invadopodia were identified by co-localization of F-actin and cortactin (white arrows). Scale bar, 10 μm. To quantify invadopodia formation, F-actin and cortactin double-positive puncta were counted from 8–10 random microscopic fields per condition. The number of invadopodia per cell was calculated by dividing the total number of invadopodia by the total number of cells. (B) Representative images and quantification of gelatin degradation in bladder cancer cells. Assessment of the functional consequences of altered invadopodia formation was investigated by fluorescent gelatin degradation assays. Areas of matrix degradation appear as dark regions beneath or within cell clusters (white arrows). Scale bar, 50 μm. Degradation was quantified from 8–10 random fields per condition. Gelatin degradation capacity of the cells was quantified by measuring the degradation area per cell and the degradation area per cell was calculated. N-numbers indicate total cell number from three independent experiments (RT4-shScr: n = 949; RT4-shp190A-1: n = 1185; RT4-shp190A-2: n = 1722; RT4-shp190A-3: n = 839; BFTC-EV: n = 702; BFTC-p190A: n = 1218; T24-EV: n = 1059 and T24- p190A: n = 934). A , B All experiments were performed at least in triplicates ( n = 3). Quantification was based on the results of three independent experiments, and results are presented as mean ± SEM. ANOVA (RT4) and Student’s t-test (BFTC, T24) were used for the statistical analyses. (* p < 0.05, ** p < 0.01).

Article Snippet: For cortactin staining, RT4, T24, BFTC cells were incubated with mouse anti-Cortactin primary antibody (1:500, sc-55579, Santa Cruz, California, USA) overnight at 4°C.

Techniques: Immunofluorescence, Functional Assay