Journal: bioRxiv

Article Title: Preimplantation factor (PIF) is an endogenous inhibitor of potassium channel K V 1.3 regulating neutrophil function during pregnancy

doi: 10.64898/2026.03.20.713251

Figure Lengend Snippet: ( A ) Human neutrophils pretreated with 300nM sPIF or vehicle (Ctrl) were introduced into E-selectin/ICAM-1/CXCL8 coated flow chambers and monitored over time (representative micrographs of neutrophils 40s, 180s and 420s after attachment, scale bar: 10µm). ( B ) Cell perimeters were analyzed over time (52 (Ctrl) and 40 (sPIF) cells from n=5 independent experiments, repeated unpaired Student’s t-tests). ( C ) sPIF and scrPIF (300nM) pretreated human neutrophils were exposed to stepwise increasing shear rates in E-selectin/ICAM-1/CXCL8-coated flow chambers and percentage of remaining neutrophils FOV −1 was quantified after each step (n=15-18 flow chambers of 8-9 independent experiments, repeated unpaired Student’s t-tests). ( D ) Schematic of experimental design of CXCL1 application during IVM of the mouse cremaster muscle. ( E ) Neutrophil adhesion was quantified before and after i.a. injection of 600ng CXCL1 in WT mice pretreated with 1µg sPIF or scrPIF (n≥5mice per group, 2-way ANOVA, Tukey’s multiple comparison). ( F-I ) β 2 integrin activation (KIM127: intermediate activation; mAB24: full activation) was assessed in isolated human neutrophils pretreated with 300nM sPIF or scrPIF and stimulated with 10nM CXCL8 and analyzed by flow cytometry ( F,H : representative flow cytometry blots, MFI: median fluorescence, n=4 independent experiments, 2-way RM ANOVA, Sidak’s multiple comparison). ( J ) Neutrophil adhesion to postcapillary venules in the cremaster muscle of WT and Kcna3 −/- i.s. injected with sPIF, scrPIF (both 1µg), PAP-1 (90µg) or vehicle 3h and stimulated i.s. with TNF 2h prior to IVM (n≥4 mice per group; 1-way ANOVA, Tukey’s multiple comparison). ( K ) Experimental design of E-selectin-triggered S100A8/A9 release assays in vitro . ( L ) S100A8/A9 levels of PBS (unstim.) or E-selectin stimulated bone marrow neutrophils from WT and Kcna3 −/- mice pretreated with sPIF, scrPIF (both 300nM) or vehicle control for 15min (n≥4mice per group; 2-way RM ANOVA; Sidak’s multiple comparison). ( M ) Schematic of ex vivo flow chamber assays. ( N ) Mean rolling velocities and ( O ) cumulative distribution of rolling velocities of neutrophils from WT mice i.p. injected with sPIF or scrPIF (both 1µg) and perfused through E-selectin and ICAM-1 coated flow chambers (n=7 mice per group; unpaired Student’s t-test; cumulative frequency: n= 109 (scrPIF) and n= 129 (sPIF) cells). ( P ) Phagocytic activity of neutrophils was assessed using fluorescent E. coli particles in human whole blood pre-incubated with sPIF (300nM) or vehicle (Ctrl) (representative micrographs, scale bar =5µm) and ( Q ) analyzed by flow cytometry (n=5 independent experiments, paired Student’s t-test). Data is presented as representative micrographs, mean±SEM, representative flow cytometry plots and as cumulative frequency.

Article Snippet: Kcna3 −/- ( Kcna3 tm1Lys ) mice were purchased from Jackson Laboratories and maintained on a C57BL/6NCrl background.

Techniques: Shear, Injection, Comparison, Activation Assay, Isolation, Flow Cytometry, Fluorescence, In Vitro, Control, Ex Vivo, Activity Assay, Incubation

Journal: Pflugers Archiv

Article Title: Just a little prick: careful cell contacts enabled by ceramic nanostraws

doi: 10.1007/s00424-026-03150-7

Figure Lengend Snippet: Protein and mRNA expression of typical ion channels in THP-1 cells. Monocytic THP-1 cells have been stimulated with PMA overnight (under exactly the same conditions as for the nanostraw experiments). After that, cells were incubated under normoxia (24 h; NOX) or under hypoxic conditions (HOX: 1% O 2 ) for 2 h, 4 h, and 24 h respectively. ( a ) mRNA expression of TRPM2 , TRPM4 and KCNA3 (encoding Kv1.3) under the given conditions. Whereas TRPM2 showed a significant downregulation after 24 h of hypoxic incubation, TRPM4 mRNA was upregulated. KCNA3 mRNA showed no changes (mean ± SD; n = 9; one way ANOVA plus Dunnett´s multiple comparisons test; ***: p < 0.001, ****: p < 0.0001). ( b ) TRPM4 and Kv1.3 protein are expressed in THP-1 cells under normoxic conditions and can be found in the cytosol and membrane of the cells (400x magnification, scale bar: 20 μm) but did not show altered expression under hypoxia (data not shown). ( c ) TRPM2 protein was cleaved under hypoxic conditions and shorter versions of the protein became detectable after only 2 h of hypoxia. 50 µg of whole cell lysate have been applied to Western blotting. Blot is representative for three independent results. ( d ) Intracellular protein distribution analysis of TRPM2 showed a prominent overall downregulation of TRPM2 after 2 h of hypoxia especially in areas close to the cell membrane. This effect seemed to recover over time (400x magnification, scale bar: 20 μm). Negative control (without primary antibody) did not show staining for TRPM2 at all

Article Snippet: The first antibody (anti-human TRPM2 from rabbit, RRID: AB_1716520, VWR International GmbH, Darmstadt, Germany, #ABNOPAB11990), anti-human TRPM4 from rabbit, (VWR International GmbH, Darmstadt, Germany, #BOSSBS-9051R, RRID: AB_3712801) or anti-human Kv1.3 from rabbit (#APC-101, RRID: AB_2040149, Alomone labs, Jerusalem, Israel) was added at a concentration of 1:100 in 3% BSA-PBS for 2 h. Cells were washed with PBS before the second antibody (goat anti-rabbit Alexa Fluor488 (#A-11008; RRID: AB_143165) for TRPM2 and goat anti-rabbit Alexa Fluor568 (#A-11011, RRID: AB_143157) for TRPM4 and Kv1.3, both from Life Technologies, Darmstadt, Germany and used in a dilution of 1:400 in PBS) was added for additional 90 min. Cover glasses were fixed with fluorescence mounting medium (#S3023, Agilent, Santa Clara, United States of America) on the slides, and cells were analyzed the following day with a fluorescence microscope (AxioVert with Axiocam 305; Zeiss, Oberkochen, Germany).

Techniques: Expressing, Incubation, Membrane, Western Blot, Negative Control, Staining