Journal: Frontiers in immunology

Article Title: Cutaneous kinase activity correlates with treatment outcomes following PI3K delta inhibition in mice with experimental pemphigoid diseases.

doi: 10.3389/fimmu.2022.865241

Figure Lengend Snippet: FIGURE 2 Parsaclisib impairs immune complex-induced neutrophil activation in vitro. Human polymorphonuclear leukocytes (PMNs) were isolated from healthy blood donors and activated by immobilized immune complexes (IC) consisting of recombinant human COL7 (hCOL7E-F) and anti- hCOL7E-F IgG1 in the presence or absence of parsaclisib at an 8-fold concentration range. Relative reactive oxygen species (ROS) release was detected by a luminescence-based assay. (A) Representative example of one donor showing the relative luminescence after IC-stimulation over the time. (B) Area under the curve (AUC, cumulative values) of luminescence, n=12. (C) PMNs were isolated from murine femurs and activated using immobilized ICs consisting of recombinant murine COL7 (mCOL7C) and anti-mCOL7C-IgG in the presence or absence of parsaclisib, n=4. (D) ROS were generated enzymatically by myeloperoxidase, catalase, and glucose oxidase, n=4. The ROS scavenger N-acetylcysteine (NAC) was used as assay control. (E) Chemotaxis of freshly isolated PMNs was induced by IL-8 in the presence of parsaclisib using a Boyden chamber assay. The attracted cell number during a time period of 60 minutes is shown as AUC. (F) Human PMNs were stimulated with immobilized ICs in the presence/absence of parsaclisib. To exclude toxicity, the amount of propidium iodide (PI)- and Annexin V-positive cells after IC stimulation was identified. (G) Cryosections of human skin were incubated with IgG isolated from EBA patients, followed by the addition of PMNs, isolated from healthy donors (cryosection assay). Split formation along the dermal epidermal junction (DEJ, dotted line) was analyzed as percentage of the whole DEJ. Asterisks indicate split formation. (A-F) Data was normalized to positive control (either IC- or IL-8-stimulated PMNs). Solvent control was always added to positive and negative controls. (B-G) Data are shown as Tukey’s box-and-whisker plots. ANOVA on ranks (Kruskal- Wallis) was applied followed by a Dunn´s multiple comparison test, (B) n=12, (C) n=2-4, (D, F) n=4, (E) n=5, (G) n=6 (for detailed information see attached raw data table), *p<0.05, **p<0.01.

Article Snippet: Frontiers in Immunology 03 Neutrophils were isolated from the cell suspension using the Neutrophil Isolation Kit, mouse, an LS Column, and a MidiMACS Separator (Miltenyi Biotec GmbH, Teterow, Germany) following the manufacturer’s protocol.

Techniques: Activation Assay, In Vitro, Isolation, Recombinant, Concentration Assay, Luminescence Assay, Generated, Control, Chemotaxis Assay, Boyden Chamber Assay, Incubation, Cryosection Assay, Positive Control, Solvent, Whisker Assay, Comparison

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Afadin is localized at cell–cell contact sites in mesangial cells and regulates migratory polarity

doi: 10.1038/labinvest.2015.133

Figure Lengend Snippet: Expression of afadin in the kidney cortex. ( a ) Sections of human kidneys were stained with anti-afadin antibody. Afadin signals were detected in glomeruli (arrow), and apical side of tubular epithelial cells (arrowheads). Scale bar, 50 μ m. ( b ) Sections of human (A and B) and rat (C and D) kidney were stained with anti-afadin antibody preadsorbed with anti-myc immunoprecipitates from cell lysates of HEK293 cells expressing myc-tagged l-afadin (A and C) or control vector (B and D). ( c ) Dual-labeling immunofluorescence of afadin (red) and ZO-1 (green) in the human kidney. Strong afadin signals were detected in the mesangium, particularly in cell–cell adhesions between mesangial cells (B, arrow) and between mesangial and endothelial cells (C, arrow). Afadin signals were also detected at cell–cell adhesions between endothelial cells of glomerular capillaries (C, arrowhead). Higher-magnification images corresponding to squares in A are shown in B and C. Scale bar, 50 μ m. ( d ) Immunogold staining of afadin detected using immunoelectron microscopy. Immunogold particles specific for afadin were detected in the mesangial cell–cell adhesion sites (arrows). Scale bars, 200 nm. M, mesangial cell.

Article Snippet: The following antibodies were obtained from commercial sources: mouse monoclonal anti-afadin antibody (BD, Franklin Lakes, NJ, USA) for immunostaining of human and rat tissue samples; rabbit polyclonal anti-Mllt4 antibody (Sigma, St Louis, MO, USA) for immunoblotting and immunostaining of cultured human mesangial cells; rabbit polyclonal anti-l-afadin antibody (Abcam, Cambridge, MA, USA) for immunoelectron microscopy; mouse monoclonal anti- β -catenin antibody (BD); mouse monoclonal anti-ZO-1 antibody (Zymed, San Francisco, CA, USA); mouse monoclonal α -SMA antibody (Progen, Heidelberg, Germany); rabbit monoclonal anti- β -actin antibody (Cell Signaling, Danvers, MA, USA); BODIPY FL phallacidin B607 (Life Technologies, Grand Island, NY, USA); mouse monoclonal anti-golgin-97 antibody (Invitrogen, Carlsbad, CA, USA) for immunostaining of cultured human mesangial cells.

Techniques: Expressing, Staining, Control, Plasmid Preparation, Labeling, Immunofluorescence, Immuno-Electron Microscopy

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Afadin is localized at cell–cell contact sites in mesangial cells and regulates migratory polarity

doi: 10.1038/labinvest.2015.133

Figure Lengend Snippet: Association of afadin and β -catenin at mesangial cell–cell contact sites. ( a ) Dual-labeling immunofluorescence of afadin and β -catenin in a rat kidney. A rat kidney section was co-stained for afadin (B, red) and β -catenin (C, green). The merged image is shown in A. Scale bar, 50 μ m. ( b ) Lysates of rat glomeruli were immunoprecipitated with rabbit IgG or rabbit anti-afadin antibody and were analyzed by western blotting with indicated antibodies. ( c ) The merged image of cultured human mesangial cells co-stained with afadin (red), β -catenin (green), and F-actin (blue). Higher-magnification images corresponding to squares in A are shown in B–E. Scale bar, 20 μ m. ( d ) Lysates of cultured human mesangial cells were immunoprecipitated with rabbit IgG or rabbit anti-afadin antibody and were analyzed by western blotting with indicated antibodies. ( e ) In situ proximity ligation assay (PLA) in cultured human mesangial cells. Rabbit anti-afadin and mouse anti- β -catenin antibodies were combined with secondary PLA probes (Olink Bioscience). The sample was co-stained with BODIPY FL phallacidin (green). The interaction events are visible as red dots at cell–cell contact sites between mesangial cells. Scale bar, 20 μ m.

Article Snippet: The following antibodies were obtained from commercial sources: mouse monoclonal anti-afadin antibody (BD, Franklin Lakes, NJ, USA) for immunostaining of human and rat tissue samples; rabbit polyclonal anti-Mllt4 antibody (Sigma, St Louis, MO, USA) for immunoblotting and immunostaining of cultured human mesangial cells; rabbit polyclonal anti-l-afadin antibody (Abcam, Cambridge, MA, USA) for immunoelectron microscopy; mouse monoclonal anti- β -catenin antibody (BD); mouse monoclonal anti-ZO-1 antibody (Zymed, San Francisco, CA, USA); mouse monoclonal α -SMA antibody (Progen, Heidelberg, Germany); rabbit monoclonal anti- β -actin antibody (Cell Signaling, Danvers, MA, USA); BODIPY FL phallacidin B607 (Life Technologies, Grand Island, NY, USA); mouse monoclonal anti-golgin-97 antibody (Invitrogen, Carlsbad, CA, USA) for immunostaining of cultured human mesangial cells.

Techniques: Labeling, Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Cell Culture, In Situ, Proximity Ligation Assay

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Afadin is localized at cell–cell contact sites in mesangial cells and regulates migratory polarity

doi: 10.1038/labinvest.2015.133

Figure Lengend Snippet: Alteration of the mesangial intercellular junction by PDGF. ( a ) The merged images of serum-starved mesangial cells stimulated with DMSO (A–C) or PDGF (50 ng/ml) for 30 min (D–F), and co-stained for afadin (red) and phallacidin (green). Higher-magnification images corresponding to squares in A and D are shown in B and C, and E and F, respectively. Scale bar, 50 μ m. ( b ) The merged images of serum-starved mesangial cells stimulated with DMSO (A–D) or PDGF (50 ng/ml) for 30 min (E–H) co-stained for afadin (red) and β -catenin (green). Higher-magnification images corresponding to squares in A and E are shown in B–D and F–H, respectively. Scale bar, 50 μ m. ( c ) Western blot analysis of the indicated antibodies in mesangal cell lysates stimulated with or without PDGF (50 ng/ml). PDGF, platelet-derived growth factor.

Article Snippet: The following antibodies were obtained from commercial sources: mouse monoclonal anti-afadin antibody (BD, Franklin Lakes, NJ, USA) for immunostaining of human and rat tissue samples; rabbit polyclonal anti-Mllt4 antibody (Sigma, St Louis, MO, USA) for immunoblotting and immunostaining of cultured human mesangial cells; rabbit polyclonal anti-l-afadin antibody (Abcam, Cambridge, MA, USA) for immunoelectron microscopy; mouse monoclonal anti- β -catenin antibody (BD); mouse monoclonal anti-ZO-1 antibody (Zymed, San Francisco, CA, USA); mouse monoclonal α -SMA antibody (Progen, Heidelberg, Germany); rabbit monoclonal anti- β -actin antibody (Cell Signaling, Danvers, MA, USA); BODIPY FL phallacidin B607 (Life Technologies, Grand Island, NY, USA); mouse monoclonal anti-golgin-97 antibody (Invitrogen, Carlsbad, CA, USA) for immunostaining of cultured human mesangial cells.

Techniques: Staining, Western Blot, Derivative Assay

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Afadin is localized at cell–cell contact sites in mesangial cells and regulates migratory polarity

doi: 10.1038/labinvest.2015.133

Figure Lengend Snippet: Decreased expression of afadin in mesangial proliferative glomerulonephritis. ( a ) Expression of afadin (A–E) and α -smooth muscle actin ( α -SMA) (F–J) before and 1, 5, 8, and 14 days after induction of rat anti-Thy-1 glomerulonephritis. The merged images are shown in K–O. Scale bar, 50 μ m. ( b ) Western blot analysis of afadin in anti-Thy-1 glomerulonephritis. Lysates of glomeruli isolated from rats before and 5, 8 and 14 days after anti-Thy-1 antibody injection were immunoblotted with indicated antibodies. The quantification of afadin in Thy-1 nephritis was shown in c . Data are shown as the mean±s.d. of three independent experiments. Statistical analysis was carried out using Student t -test. * P <0.005. ( d ) Afadin expression in human glomerulonephritis. Double immunostaining for afadin (red) and zonula occludens-1 (ZO-1, green) in a control case (A) and patients with IgA nephropathy (B–F) and membranoproliferative glomerulonephritis (MPGN) (G and H) are shown. Higher-magnification images corresponding to squares in A through H are shown in I through P, respectively. Scale bar, 50 μ m.

Article Snippet: The following antibodies were obtained from commercial sources: mouse monoclonal anti-afadin antibody (BD, Franklin Lakes, NJ, USA) for immunostaining of human and rat tissue samples; rabbit polyclonal anti-Mllt4 antibody (Sigma, St Louis, MO, USA) for immunoblotting and immunostaining of cultured human mesangial cells; rabbit polyclonal anti-l-afadin antibody (Abcam, Cambridge, MA, USA) for immunoelectron microscopy; mouse monoclonal anti- β -catenin antibody (BD); mouse monoclonal anti-ZO-1 antibody (Zymed, San Francisco, CA, USA); mouse monoclonal α -SMA antibody (Progen, Heidelberg, Germany); rabbit monoclonal anti- β -actin antibody (Cell Signaling, Danvers, MA, USA); BODIPY FL phallacidin B607 (Life Technologies, Grand Island, NY, USA); mouse monoclonal anti-golgin-97 antibody (Invitrogen, Carlsbad, CA, USA) for immunostaining of cultured human mesangial cells.

Techniques: Expressing, Western Blot, Isolation, Injection, Double Immunostaining, Control

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Afadin is localized at cell–cell contact sites in mesangial cells and regulates migratory polarity

doi: 10.1038/labinvest.2015.133

Figure Lengend Snippet: Impaired formation of front-rear polarity in afadin-depleted mesangial cells. ( a ) Confluent human mesangial cell monolayers of control siRNA and siRNAs #1 and #2 for afadin were manually scratched and cultured for 24 h. Cells were stained with goldin97 (golgi complex marker, red) and DAPI (blue). The line indicates the leading edge of the wound. ( b ) The percentage of Golgi apparatus facing the wound, facing the other direction, and non-polarized with respect to the wound was calculated as described in the Materials and Methods. * P <0.05 using Student's t -test. Data are shown as the mean±s.d. of three independent experiments.

Article Snippet: The following antibodies were obtained from commercial sources: mouse monoclonal anti-afadin antibody (BD, Franklin Lakes, NJ, USA) for immunostaining of human and rat tissue samples; rabbit polyclonal anti-Mllt4 antibody (Sigma, St Louis, MO, USA) for immunoblotting and immunostaining of cultured human mesangial cells; rabbit polyclonal anti-l-afadin antibody (Abcam, Cambridge, MA, USA) for immunoelectron microscopy; mouse monoclonal anti- β -catenin antibody (BD); mouse monoclonal anti-ZO-1 antibody (Zymed, San Francisco, CA, USA); mouse monoclonal α -SMA antibody (Progen, Heidelberg, Germany); rabbit monoclonal anti- β -actin antibody (Cell Signaling, Danvers, MA, USA); BODIPY FL phallacidin B607 (Life Technologies, Grand Island, NY, USA); mouse monoclonal anti-golgin-97 antibody (Invitrogen, Carlsbad, CA, USA) for immunostaining of cultured human mesangial cells.

Techniques: Control, Cell Culture, Staining, Marker

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: Afadin is localized at cell–cell contact sites in mesangial cells and regulates migratory polarity

doi: 10.1038/labinvest.2015.133

Figure Lengend Snippet: Impairment of directional mesangial cell migration by afadin depletion. ( a ) Confluent mesangial cell monolayers were manually scratched and cultured for 24 h. Light-microscopy images of confluent human mesangial cell monolayers of control siRNA and siRNAs #1 and #2 for afadin immediately after wounding (0 h) and 24 h later. ( b ) The percent of initial wound area was calculated as described in the Materials and Methods. P <0.05 using Student's t -test. Data are shown as the mean±s.d. of three independent experiments. ( c ) Three days after transfection with control siRNA or afadin siRNAs, mesangial cells were subjected to a modified Boyden chamber assay with or without PDGF (30 ng/ml) in the lower chamber. * P <0.05 using Student's t- test. Data are shown as the mean±s.d. of three independent experiments.

Article Snippet: The following antibodies were obtained from commercial sources: mouse monoclonal anti-afadin antibody (BD, Franklin Lakes, NJ, USA) for immunostaining of human and rat tissue samples; rabbit polyclonal anti-Mllt4 antibody (Sigma, St Louis, MO, USA) for immunoblotting and immunostaining of cultured human mesangial cells; rabbit polyclonal anti-l-afadin antibody (Abcam, Cambridge, MA, USA) for immunoelectron microscopy; mouse monoclonal anti- β -catenin antibody (BD); mouse monoclonal anti-ZO-1 antibody (Zymed, San Francisco, CA, USA); mouse monoclonal α -SMA antibody (Progen, Heidelberg, Germany); rabbit monoclonal anti- β -actin antibody (Cell Signaling, Danvers, MA, USA); BODIPY FL phallacidin B607 (Life Technologies, Grand Island, NY, USA); mouse monoclonal anti-golgin-97 antibody (Invitrogen, Carlsbad, CA, USA) for immunostaining of cultured human mesangial cells.

Techniques: Migration, Cell Culture, Light Microscopy, Control, Transfection, Modification, Boyden Chamber Assay

Journal: Yonsei Medical Journal

Article Title: Heat Shock Factor 1 Predicts Poor Prognosis of Gastric Cancer

doi: 10.3349/ymj.2018.59.9.1041

Figure Lengend Snippet: Downregulation of HSF1 expression reduces proliferation, migration, and invasion of gastric cancer cells. AGS and MKN28 cells were transfected with a scrambled siRNA (scRNA) or two small-interfering RNAs (siRNAs) specific for HSF1 (siRNA #1 and #2). (A) HSF1 protein expression was detected by Western blot analysis. β-actin was used as a loading control. (B) WST assay was performed to detect cell viability. (C and D) Transwell assays to evaluate (C) migration and (D) invasion of cells (×200). The histogram is represented as mean±SEM (n=3). p values were calculated using ANOVA and statistically significant differences are indicated as * ( * p <0.001). HSF1, heat shock factor 1.

Article Snippet: After 24-hour transfection, 1×10 4 cells from each well were isolated and added to the upper transwell chamber (Corning Costar, Tewksbury, MA, USA) that carried a filter coated with 0.5 mg/mL of collagen type I (BD Biosciences, Seoul, Korea) for the migration assay or a filter coated with Matrigel (1:15) (BD Biosciences) for the invasion assay.

Techniques: Expressing, Migration, Transfection, Western Blot, Control, WST Assay

Journal: Yonsei Medical Journal

Article Title: Heat Shock Factor 1 Predicts Poor Prognosis of Gastric Cancer

doi: 10.3349/ymj.2018.59.9.1041

Figure Lengend Snippet: Overexpression of HSF1 promotes proliferation, migration, and invasion of gastric cancer cells. Overexpression of HSF1 in AGS and MKN28 cells was achieved by transfection with an empty vector (pcDNA_EV) or HSF1 overexpression vector (pcDNA_HSF1). (A) Expressions of HSF1 mRNA and protein were detected by Western blot analysis. β-actin was used as a loading control. (B) WST assay was performed to detect cell viability. (C and D) Transwell migration assays to evaluate (C) migration and (D) invasion activity of cells (×200). Data are represented as mean±SEM (n=3). p values were calculated using Student's t-test ( * p <0.001, † p =0.001). HSF1, heat shock factor 1.

Article Snippet: After 24-hour transfection, 1×10 4 cells from each well were isolated and added to the upper transwell chamber (Corning Costar, Tewksbury, MA, USA) that carried a filter coated with 0.5 mg/mL of collagen type I (BD Biosciences, Seoul, Korea) for the migration assay or a filter coated with Matrigel (1:15) (BD Biosciences) for the invasion assay.

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Western Blot, Control, WST Assay, Activity Assay