Journal: Frontiers in Microbiology

Article Title: Afadin Downregulation by Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Gastric Cells

doi: 10.3389/fmicb.2018.02712

Figure Lengend Snippet: Impact of H. pylori infection on Afadin expression. MKN74 and NCI-N87 gastric cell lines were left untreated or infected with H. pylori 26695 (A,B) or infected with H. pylori 60190, or with the respective CagA - , CagE - and VacA - mutants (C) , for 24 h at a MOI of 100. Afadin detection by immunofluorescence (red) and quantification of the fluorescence intensity at the membrane and the nucleus (A) , and by Western blot and its quantification, using GAPDH or tubulin as loading controls (B,C) . Scale bar, 10 μm. Data correspond to the mean value ± SEM and are representative of at least three independent experiments. Statistical significance was evaluated with the Student’s t- test or with one-way ANOVA.

Article Snippet: An siRNA silencing Afadin expression (sc-43007, Santa Cruz) and a negative control siRNA (All Stars Negative Control siRNA; QIAGEN, Germany), were used at a final concentration of 75 nM in serum- and antibiotic-free Opti-MEM medium (Invitrogen).

Techniques: Infection, Expressing, Immunofluorescence, Fluorescence, Membrane, Western Blot

Journal: Frontiers in Microbiology

Article Title: Afadin Downregulation by Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Gastric Cells

doi: 10.3389/fmicb.2018.02712

Figure Lengend Snippet: Afadin downregulation displaces apical junctional complex proteins and promotes the formation of actin stress fibers. Double immunofluorescence of Afadin (red) with AJs proteins E-cadherin and β-catenin (green), and with TJs proteins ZO-1 and occludin (green) in MKN74 cells transfected with a non-silencing siRNA (siNS) or with a siRNA to Afadin (siAFDN). Immunofluorescence of actin (green) and of Snail (red) are also shown. Nuclei were counterstained with DAPI. Scale bar, 10 μm. White arrows represent cells with Afadin not efficiently silenced by the siRNA and that retain the epithelial morphology; Yellow arrows, lamellipodia; cyan arrows, filopodia (A) . Quantification of Afadin fluorescence intensity in MKN74 cells in both membrane and the nucleus upon treatment with non-silencing siRNA or with a siRNA to Afadin (B) .

Article Snippet: An siRNA silencing Afadin expression (sc-43007, Santa Cruz) and a negative control siRNA (All Stars Negative Control siRNA; QIAGEN, Germany), were used at a final concentration of 75 nM in serum- and antibiotic-free Opti-MEM medium (Invitrogen).

Techniques: Immunofluorescence, Transfection, Fluorescence, Membrane

Journal: Frontiers in Microbiology

Article Title: Afadin Downregulation by Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Gastric Cells

doi: 10.3389/fmicb.2018.02712

Figure Lengend Snippet: Afadin downregulation does not alter the expression of apical junctional complex proteins, but upregulates the EMT marker Snail. Western blot analyses of MKN74 and NCI-N87 cells, cells transfected with a non-silencing siRNA (siNS), or with a siRNA to Afadin (siAFDN), evaluating (A) apical junctional complex proteins E-cadherin, β-catenin, ZO-1, and occludin, and (B) mesenchymal markers Snail, N-cadherin, ZEB1, Slug, and Vimentin. GAPDH was used as loading control. Numbers below the bands correspond to quantifications. Data are representative of three independent experiments. Statistical significance was evaluated with the Student’s t- test.

Article Snippet: An siRNA silencing Afadin expression (sc-43007, Santa Cruz) and a negative control siRNA (All Stars Negative Control siRNA; QIAGEN, Germany), were used at a final concentration of 75 nM in serum- and antibiotic-free Opti-MEM medium (Invitrogen).

Techniques: Expressing, Marker, Western Blot, Transfection, Control

Journal: Frontiers in Microbiology

Article Title: Afadin Downregulation by Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Gastric Cells

doi: 10.3389/fmicb.2018.02712

Figure Lengend Snippet: Afadin downregulation alters the TJ barrier function and increases cell motility and invasion. (A) Transepithelial electrical resistance (TER) measurements of MKN74 gastric cells during a period of 6 days post Afadin silencing (siAFDN) in comparison with non-silenced (siNS) and with untreated cells, and during 2 days after H. pylori infection in comparison with uninfected cells. (B) Cell monolayer permeability to 4 kDa FITC-Dextran on day 6 post Afadin silencing and on day 2 after H. pylori infection. (C–F) Single cell motility analysis: (C) Time-lapse microscopy photos of cells during the 24 h period. (D) Representative graphs of the X and Y coordinates (in pixels, px) of each cell path, on a fibronectin coated surface. (E) Cell velocity in μm/minute and (F) total distance covered by and individual cell in μm. (G,H) Cell invasion capacity on Matrigel-coated transwells for MKN74 cells transfected with siNS control or transfected with siAFDN (G) , and for MKN74 cells infected or not with H. pylori 26695 (H) . Data are presented as mean ± SEM. Statistical significance was evaluated with the one- and two-way ANOVA and with Student’s t -test.

Article Snippet: An siRNA silencing Afadin expression (sc-43007, Santa Cruz) and a negative control siRNA (All Stars Negative Control siRNA; QIAGEN, Germany), were used at a final concentration of 75 nM in serum- and antibiotic-free Opti-MEM medium (Invitrogen).

Techniques: Comparison, Infection, Permeability, Time-lapse Microscopy, Transfection, Control

Journal: Frontiers in Microbiology

Article Title: Afadin Downregulation by Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Gastric Cells

doi: 10.3389/fmicb.2018.02712

Figure Lengend Snippet: H. pylori infection affects the expression and localization of the Afadin protein in the human gastric mucosa. (A) AFDN gene expression in the stomach of 181 (114 uninfected and 67 H. pylori -infected) individuals without disease, retrieved from the GTEx database. RPKM, reads per kilobase million. Significance was evaluated with Kruskal–Wallis statistics. (B–F) Immunohistochemical detection of Afadin in paraffin-embedded sections of the gastric mucosa of individuals uninfected ( n = 20) or infected with H. pylori ( n = 22). (B) Graphical representation of Afadin alterations in the two biological groups. Statistical significance was determined with the Fisher’s exact test. Representative micrographs of Afadin staining in an uninfected individual, showing a strong intensity at the apicolateral epithelial cell–cell contacts (black arrowheads) (C) , and a belt-like pattern (red arrowheads) (D) in longitudinal and cross sections, respectively, and in H. pylori -infected individuals, presenting decreased intensity/loss of membrane staining at the cell–cell contacts (blue arrowheads) (E) or showing cytoplasmic staining in areas of epithelial tufting (F) .

Article Snippet: An siRNA silencing Afadin expression (sc-43007, Santa Cruz) and a negative control siRNA (All Stars Negative Control siRNA; QIAGEN, Germany), were used at a final concentration of 75 nM in serum- and antibiotic-free Opti-MEM medium (Invitrogen).

Techniques: Infection, Expressing, Gene Expression, Immunohistochemical staining, Staining, Membrane

Journal: Oncogene

Article Title: EphA2 regulates vascular permeability and prostate cancer metastasis via modulation of cell junction protein phosphorylation

doi: 10.1038/s41388-024-03206-x

Figure Lengend Snippet: a , b Proteome Profiler Human Phospho-Kinase Array and western blot analysis validating pathway regulation downstream of EphA2 kinase activation in PC-3 cells. PC-3 cells were left untreated or incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (A1) for 20 min to activate the EphA2 receptor. Representative images of array and western blot membranes are shown. β-actin is the western blot loading control. Increased PARD3 tyrosine phosphorylation was shown by immunoprecipitating PARD3 and subsequent probing with an anti-phospho-tyrosine antibody. Bar graph shows the quantification of the Proteome Profiler array as log2(fold change (efnA1-Fc:Fc control)). Mean ± SE, ( n = 4 biological replicates). c Western blot analysis assessing EphA2-signaling in a panel of prostate, breast, colon and brain cancer cell lines. Cell lines were incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (A1) for 20 min. SHB phosphorylation on Y246 is consistently upregulated by EphA2 receptor activation across all tested cell lines. Downregulation of Afadin (S1718/S1799) and NDRG1 was observed only in cell lines which responded with a robust downregulation of Akt phosphorylation on S473 (PC-3, MDA-MB-231, BT-519, HCT-116 and MN1).

Article Snippet: Briefly, coverslips were blocked with Duolink Blocking Solution for 60 min, then incubated for 60 min with the anti-afadin antibody (Novus Biochemicals #NBP1-90219) and anti-EphA2 1F7 mAb (in-house), both diluted 1:50 in Duolink Antibody Diluent.

Techniques: Western Blot, Activation Assay, Incubation, Control, Phospho-proteomics

Journal: Oncogene

Article Title: EphA2 regulates vascular permeability and prostate cancer metastasis via modulation of cell junction protein phosphorylation

doi: 10.1038/s41388-024-03206-x

Figure Lengend Snippet: a Western blot analysis of the effect of transient SHB siRNA knockdown on Akt and Erk pathway inhibition in response to EphA2 activation. Cell lines were incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (efnA1-Fc) for 20 min. A representative western botting image is shown. β-actin is the loading control. Relative phosphorylated protein levels of phospho-EphA, phospho-Erk and phospho-AKT were quantified and are shown as bar charts. Mean ± SE ( n = 3 biological replicates shown as individual data points). SHB Knockdown was confirmed by quantitative QPCR and relative mRNA expression levels determined using the 2 −ΔΔCt method with β-actin as the housekeeping gene and the Lipofectamine control as the reference sample. Mean ± SE ( n = 3 biological replicates). Knockdown of SHB diminished the ligand-induced EphA2-mediated Erk1/2 pathway inhibition, while the inhibitory effect on the Akt pathway remained intact. b Representative confocal immunofluorescence images of afadin (AFDN, red) and EphA2 (green) cellular localization in response to EphA2 stimulation with clustered ephrin-A1-Fc (efnA1-Fc). EphA2 activation regulates the intracellular localization of afadin. Cell nuclei are labeled with DAPI (blue). Scale bars are 20 µm. c Transient knockdown of afadin (AFDN) with two different siRNA sequences was confirmed by western blot analysis. β-actin is the loading control. Bar chart shows knockdown efficiency as determined by densitometry of western blotting results. Mean ± SE ( n = 3 biological replicates). *** p value < 0.001, ns. not significant (One-way ANOVA with post-hoc Šidák test for multiple pairwise comparisons). d Transwell invasion assay comparing invasion of afadin (AFDN) knockdown cells compared to untransfected and negative control siRNA-transfected PC-3 cells. Mean ± SE ( n = 5 biological replicates). * p value < 0.05, ns. not significant (One-way ANOVA with post-hoc Šidák test for multiple pairwise comparisons).

Article Snippet: Briefly, coverslips were blocked with Duolink Blocking Solution for 60 min, then incubated for 60 min with the anti-afadin antibody (Novus Biochemicals #NBP1-90219) and anti-EphA2 1F7 mAb (in-house), both diluted 1:50 in Duolink Antibody Diluent.

Techniques: Western Blot, Knockdown, Inhibition, Activation Assay, Incubation, Control, Expressing, Immunofluorescence, Labeling, Transwell Invasion Assay, Negative Control, Transfection

Journal: Nature Communications

Article Title: Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions

doi: 10.1038/s41467-021-25011-w

Figure Lengend Snippet: List of SILAC-enriched PAK4-proximal proteins.

Article Snippet: Primary antibodies were obtained from the following sources: rabbit PAK4 (Proteintech 14685-1-AP); mouse p120-catenin (Santa Cruz sc-23873); rabbit β-catenin (Cell Signaling #9582S); mouse β-catenin (Santa Cruz sc-7963); rabbit Afadin (Sigma A0224); mouse Afadin (R&D Systems MAB78291); rat ZO-1 (Santa Cruz sc-33725); rabbit LZTS2 (Proteintech 15677-1-AP); rabbit DLG5 (Abcam ab86783), rabbit Scribble (Santa Cruz sc-28737), rabbit p120-catenin pS320 (Cell Signaling #8016S), mouse β-tubulin (Sigma T4026).

Techniques: Multiplex sample analysis, Binding Assay

Journal: Nature Communications

Article Title: Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions

doi: 10.1038/s41467-021-25011-w

Figure Lengend Snippet: a Confluent MDCK cells grown on glass coverslips (4 days) were fixed with methanol and co-stained using rabbit anti-PAK4 and mouse anti-Afadin/anti-β-catenin or anti-ZO1. Images were collected on an Olympus Fluoview confocal microscope with ×100 oil objective. Lower panels show MDCK cell grown in Matrigel to form 3D-cultured acini (8 days) fixed in methanol and immuno-stained with anti-PAK4, anti-p120ctn (×60 objective). b Sub-confluent (2 day) cells were stained using rabbit antibodies specific for PAK4 and mouse antibody specific for p120 (×60 objective). Repeated in three independent experiments. Scale bars: 10 μm.

Article Snippet: Primary antibodies were obtained from the following sources: rabbit PAK4 (Proteintech 14685-1-AP); mouse p120-catenin (Santa Cruz sc-23873); rabbit β-catenin (Cell Signaling #9582S); mouse β-catenin (Santa Cruz sc-7963); rabbit Afadin (Sigma A0224); mouse Afadin (R&D Systems MAB78291); rat ZO-1 (Santa Cruz sc-33725); rabbit LZTS2 (Proteintech 15677-1-AP); rabbit DLG5 (Abcam ab86783), rabbit Scribble (Santa Cruz sc-28737), rabbit p120-catenin pS320 (Cell Signaling #8016S), mouse β-tubulin (Sigma T4026).

Techniques: Staining, Microscopy, Cell Culture

Journal: Nature Communications

Article Title: Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions

doi: 10.1038/s41467-021-25011-w

Figure Lengend Snippet: a Confluent U2OS cells grown on glass coverslips for 3 days then fixed with methanol and immuno-stained for PAK4 and Afadin or β-catenin antibodies as described in 'Methods'. Note the concordance in localization (top panel) between PAK4 and Afadin compared with that of β-catenin. Images were collected on an Olympus Fluoview confocal microscope with ×100 oil objective. b Cells were similarly stained using anti-LZTS2 (rabbit) and mouse anti-Afadin, or anti-β-catenin. Again note the concordance of staining of LZTS2 with Afadin. Repeated in three independent experiments. Scale bars: 10 μm.

Article Snippet: Primary antibodies were obtained from the following sources: rabbit PAK4 (Proteintech 14685-1-AP); mouse p120-catenin (Santa Cruz sc-23873); rabbit β-catenin (Cell Signaling #9582S); mouse β-catenin (Santa Cruz sc-7963); rabbit Afadin (Sigma A0224); mouse Afadin (R&D Systems MAB78291); rat ZO-1 (Santa Cruz sc-33725); rabbit LZTS2 (Proteintech 15677-1-AP); rabbit DLG5 (Abcam ab86783), rabbit Scribble (Santa Cruz sc-28737), rabbit p120-catenin pS320 (Cell Signaling #8016S), mouse β-tubulin (Sigma T4026).

Techniques: Staining, Microscopy

Journal: Nature Communications

Article Title: Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions

doi: 10.1038/s41467-021-25011-w

Figure Lengend Snippet: The sub-apical complex includes a structure recently described as well the tight junction (TJ) and region ‘below’ this typically described adherens junction (AJ), which includes both Afadin and cadherin complexes. The smaller cadherin punctate junctions along the lateral contacts are not explicitly indicated. Typical non-transmembrane components (for example, p120ctn and β-catenin) are often used as markers and these are indicative of well-studied components. The Afadin/nectin compartment in vertebrates is often spatially segregated from cadherin as described in the text.

Article Snippet: Primary antibodies were obtained from the following sources: rabbit PAK4 (Proteintech 14685-1-AP); mouse p120-catenin (Santa Cruz sc-23873); rabbit β-catenin (Cell Signaling #9582S); mouse β-catenin (Santa Cruz sc-7963); rabbit Afadin (Sigma A0224); mouse Afadin (R&D Systems MAB78291); rat ZO-1 (Santa Cruz sc-33725); rabbit LZTS2 (Proteintech 15677-1-AP); rabbit DLG5 (Abcam ab86783), rabbit Scribble (Santa Cruz sc-28737), rabbit p120-catenin pS320 (Cell Signaling #8016S), mouse β-tubulin (Sigma T4026).

Techniques:

Journal: Nature Communications

Article Title: Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions

doi: 10.1038/s41467-021-25011-w

Figure Lengend Snippet: a To assess the effect of single amino-acid substitution on a selected optimal PAK4 substrate, we tested 13aa synthetic peptides (Pepspots, Jerini) derived from PAK4 pseudosubstrate motif (SARRPKPLVDPAD) in which the proline in bold is replaced by Ser(0). This is similar to an optimal substrate for PAKs (RKRRNSLAYKK) termed PAKtide but optimal for kinase binding. Based on structural considerations the Arg side chain at position −2 or −3 occupies a pocket that mediates interactions found in PAKs and other S/T kinases, including PKA . The contribution of each side-chain to peptide phosphorylation was assessed by sequential alanine substitution. b We selected in vivo basic-directed phosphorylation sites identified in Afadin, scribble, ZO-1, DLG5 and p120ctn as compiled in the Phosphosite database (V6.5.9.3). The corresponding synthetic peptides were synthesized and subjected to in situ phosphorylation. The extent of phosphorylation (32P signal) ranges from detectable (−/+) to very strong (+++), with no signal shown as ns. c Schematic of the domain structure and relative positions of the p120-catenin phosphorylation sites as indicated in the table. d Western blot showing the inhibition of p120ctn Ser320 phosphorylation by PF-3758309 U2OS cells .

Article Snippet: Primary antibodies were obtained from the following sources: rabbit PAK4 (Proteintech 14685-1-AP); mouse p120-catenin (Santa Cruz sc-23873); rabbit β-catenin (Cell Signaling #9582S); mouse β-catenin (Santa Cruz sc-7963); rabbit Afadin (Sigma A0224); mouse Afadin (R&D Systems MAB78291); rat ZO-1 (Santa Cruz sc-33725); rabbit LZTS2 (Proteintech 15677-1-AP); rabbit DLG5 (Abcam ab86783), rabbit Scribble (Santa Cruz sc-28737), rabbit p120-catenin pS320 (Cell Signaling #8016S), mouse β-tubulin (Sigma T4026).

Techniques: Derivative Assay, Binding Assay, Phospho-proteomics, In Vivo, Synthesized, In Situ, Western Blot, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: In Vivo Target Engagement Assessment of Nintedanib in a Double-Hit Bleomycin Lung Fibrosis Rat Model

doi: 10.3390/ijms27010064

Figure Lengend Snippet: Assessment of Nintedanib target engagement. Nintedanib (light blue columns) did not modify lung levels of PDGF ( A ), which was unaffected by BLM, while significantly attenuated FGF2 lung levels ( B ) were enhanced by BLM. In contrast, lung ( C ) and plasma ( D ) VEGF levels were significantly increased by Nintedanib treatment relative to both untreated (control) and BLM treated groups. Panel ( E , F ) illustrates the rise in VEGF levels in lung and plasma, respectively, observed at different time points following Nintedanib treatment in BLM-free animals. Statistical analysis was performed with the one-way ANOVA followed by Dunnett’s test. A p -value < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01 when compared to the control group (cream columns, Panels A – D ) or time points 0 (Panels E , F ). # p < 0.05 when compared to BLM + Vehicle group (teal blue columns, Panels A – D ).

Article Snippet: All markers were quantified by Enzyme-linked immunosorbent assays (ELISA) commercial kits: procollagen-I (ab210579, Abcam, Cambridge, UK), WISP-1 (Mouse/Rat WISP-1/CCN4 Quantikine ELISA Kit, MWSP10, Bio-Techne, Minneapolis, MN, USA), MMP7 (Rat MMP-7 ELISA Kit, NBP3-06896, Bio-Techne, Minneapolis, MN, USA), PDGF (Rat PDGF ELISA Kit, orb567597, Biorbyt Cambridge, UK), FGF2 (Mouse and Rat FGF basic/FGF2/bFGF ELISA Kit—Quantikine, MFB00, Bio-Techne, Minneapolis, MN, USA) and VEGF-A (Rat VEGF-A ELISA Kit, ab100787, Abcam, Cambridge, UK).

Techniques: Drug discovery, Clinical Proteomics, Control, Cream

Journal: International Journal of Molecular Sciences

Article Title: In Vivo Target Engagement Assessment of Nintedanib in a Double-Hit Bleomycin Lung Fibrosis Rat Model

doi: 10.3390/ijms27010064

Figure Lengend Snippet: Down-regulated pathways derived from phosphorylated proteins. The down-regulated proteins enrich pathways associated with VEGF, PDGF, and FGF, mainly 3 h after the administration. The blue shades indicate the q-value.

Article Snippet: All markers were quantified by Enzyme-linked immunosorbent assays (ELISA) commercial kits: procollagen-I (ab210579, Abcam, Cambridge, UK), WISP-1 (Mouse/Rat WISP-1/CCN4 Quantikine ELISA Kit, MWSP10, Bio-Techne, Minneapolis, MN, USA), MMP7 (Rat MMP-7 ELISA Kit, NBP3-06896, Bio-Techne, Minneapolis, MN, USA), PDGF (Rat PDGF ELISA Kit, orb567597, Biorbyt Cambridge, UK), FGF2 (Mouse and Rat FGF basic/FGF2/bFGF ELISA Kit—Quantikine, MFB00, Bio-Techne, Minneapolis, MN, USA) and VEGF-A (Rat VEGF-A ELISA Kit, ab100787, Abcam, Cambridge, UK).

Techniques: Derivative Assay