Journal: International Journal of Molecular Sciences

Article Title: Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells

doi: 10.3390/ijms21020527

Figure Lengend Snippet: Temporal redistribution of TJ proteins and barrier dysfunction caused by EPEC. ( A–C ) SKCO-15 cells were plated on Transwells and infected or not (UI) with EPEC. Total JAM-A, JAM-A S285, JAM-A Y280, occludin, and ZO-1 localization and TER were determined. ( A ) EPEC does not alter the distribution of total JAM-A. In contrast, JAM-A S285 is displaced from the cell–cell contacts to the cytoplasm at 30 min post-infection. Tyrosine phosphorylation of JAM-A Y280 is apparent at 60–120 min post-infection. Scale bars, 10 µm. ( B ) EPEC induces the endocytosis of occludin and ZO-1 at 1 and 2 h post-infection, respectively. Scale bars, 10 µm. ( C ) TER drops significantly as early as 45 min post-infection and progressively decreases over time as more TJ proteins are displaced. TER reported as percent change from baseline. * p < 0.01, *** p < 0.001.

Article Snippet: Par3 (07-330, EMD Millipore), Par6 (ab49776 and ab6022, Abcam, Cambridge, MA, USA), aPKCζ (sc-17781, Santa Cruz Biotechnology, Dallas, TX, USA), p-aPKCζ–T560 (ab62372, Abcam, Cambridge, MA, USA), p-aPKCζ–T410 (sc-12894R, Santa Cruz Biotechnology, Dallas, TX, USA), actin (A2066, Sigma-Aldrich, St. Louis, MO, USA), F-actin BODIPY 558/568 Phalloidin (B3475, Invitrogen, Life Technologies Carlsbad, CA, USA), occludin (33-1500, Invitrogen, Life Technologies, Carlsbad, CA, USA), JAM-A S285 (sc-17430, Santa Cruz Biotechnology, Dallas, TX, USA), JAM-A Y280 (600-401-GN5, Rockland Immunochemicals Inc, Limerick, PA), JAM-A (361700, Invitrogen, Life Technologies, Carlsbad, CA, USA), and ZO-1 (61-7300, Invitrogen, Life Technologies, Carlsbad, CA, USA).

Techniques: Infection, Phospho-proteomics

Journal: Frontiers in Cardiovascular Medicine

Article Title: Differential Induction of the ADAM17 Regulators iRhom1 and 2 in Endothelial Cells

doi: 10.3389/fcvm.2020.610344

Figure Lengend Snippet: Shear stress- and TNFα-mediated induction of iRhom1 and iRhom2 lead to increased active ADAM17 on the cell surface. HUVECs were cultured for 24 h under static conditions or with a shear stress of 30 dyn/cm 2 and subsequently stimulated with or without 10 ng/ml TNFα for another 24 h with or without shear stress. Cells were then analyzed for ADAM17 protein expression (A–C) and ADAM17 surface expression (D,E) . The concentrated supernatant was analyzed for levels of soluble JAM-A (F) . (A–C) Western blot results are shown as the ratio of the densitometric signal of total ADAM17 (tADAM17) and GAPDH (A) , as the ratio of the densitometric signal of mature ADAM17 (mADAM17) and pro ADAM17 (pADAM17) (B) , and as representative blot (C) . (D,E) Results of the flow cytometric analysis are shown as geometric mean of the fluorescence intensity representing the relative ADAM17 surface expression (D) and as representative histogram (E) . (F) Results of the JAM-A ELISA are presented as concentration of soluble JAM-A in pg/ml. Four independent experiments were performed with HUVECs from four different donors. Data are shown as mean + standard deviation (SD) and as black and gray dots representing the individual data points. Statistical differences to the corresponding static control are indicated by asterisks (* p < 0.05, ** p < 0.01, and *** p < 0.001) and significant differences between untreated control cells and cells treated with TNFα are indicated as hashes ( # p < 0.05, ## p < 0.01, and ### p < 0.001).

Article Snippet: The ELISA was performed according to manufacturer's instructions (JAM-A ELISA kit, SinoBiological, Beijing, China).

Techniques: Cell Culture, Expressing, Western Blot, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation

Journal: Cell reports

Article Title: SerpinB3 drives cancer stem cell survival in glioblastoma

doi: 10.1016/j.celrep.2022.111348

Figure Lengend Snippet: (A) Graphical abstract of His-JAM-A pulldown and liquid chromatography-mass spectrometry (LC-MS) procedure. (B) Verification of LC-MS results by western blot. His-tagged JAM-A was overexpressed in T4121 cancer stem cells (CSCs), and protein was isolated and mixed with nickel beads. The bound fraction was subjected to immunoblotting with antibodies to SerpinB3 and JAM-A. (C) Immunofluorescent staining demonstrating co-expression of JAM-A and SerpinB3 in T387 PDX glioblastoma tumor model (scale bar, 10 μm). (D) JAM-A was knocked down in T4121 CSCs with 2 separate shRNA constructs, and SerpinB3 expression was measured. Actin was used as a loading control in this and all subsequent western blots. (E) T387 and T4121 CSCs expressing JAM-A KD2 shRNA or NT control were treated with cycloheximide, and SerpinB3 expression was measured at 6 and 12 h post-treatment. (F) Western blot demonstrating knockdown of SerpinB3 with each shRNA, KD1, and KD2. (G) Fold change in cell viability at day 7, normalized to day 0, in 3 PDX glioblastoma models. Cell viability measured with CellTiter-Glo Luminescent Cell Viability Assay (5 technical replicates per condition, per tumor model). (H and I) Kaplan-Meier curves depicting survival of mice with 20,000 T4121 or T387 tumor cells intracranially injected. Cells were transfected with either non-target (SHC002) or SerpinB3 (KD1 or KD2) shRNA, with n = 10 mice per group. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by 1-way ANOVA with Dunnett’s multiple comparisons test or log rank test for survival data. Error bars represent standard deviations.

Article Snippet: N-terminal His-tagged full-length JAM-A , Sinobiologicals , HG10198-NH.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Isolation, Staining, Expressing, shRNA, Construct, Cell Viability Assay, Injection, Transfection