Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) Domain structure of GDE2, GDE3 and GDE6 (left panel), and the transmembrane scheme of GDE3 (right panel). GDPD denotes catalytic glycerophosphodiesterase domain. Asterisk in GDPD domain depicts catalytic His residue in both GDE2 and GDE3. ( B ) Immunoblot analysis of uPAR release into the medium. HEK-uPAR cells transfected with empty vector (control), GDE2 or GDE3. PI-PLC served as positive control. ( C ) Mutant GDE3(H229A) fails to release uPAR. ( D ) Partial loss of uPAR from the plasma membrane by GDE3, as measured by flow cytometry. ( E ) TIRF microscopy reveals loss of uPAR from the basolateral plasma membrane. Box plot shows uPAR-GFP intensity at the ventral membrane (n = 3, mean ±SEM ****p<0001). ( F ) Homology modeling of the GDE2 and GDE3 catalytic domains showing surface charge distributions (blue, positive; red, negative; green line, putative GPI-binding groove; yellow line, proposed substrate-binding surface). The active site is indicated by glycerol-3-phosphate located at the template structure.

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Residue, Western Blot, Transfection, Plasmid Preparation, Control, Positive Control, Mutagenesis, Clinical Proteomics, Membrane, Flow Cytometry, Microscopy, Binding Assay

Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) Confocal images of HEK293 cells expressing human GDE2-HA, GDE3-HA or GDE6-HA, as indicated; bar, 10 μm. ( B ) GDE3 localizes to distinct microdomains (yellow square, open arrows) and filopodia-like extensions (orange square, solid arrow), as visualized by confocal and super-resolution microscopy. Bars, 10 μm and 1 μm respectively. ( C ) Immunoblot analysis showing that GDE3 reduces the membrane-anchored uPAR pool and competes with PI-PLC. The medium and lysates of HEK-uPAR cells expressing GDE3 or empty vector (EV) were analyzed without or with PI-PLC treatment (45 min.) of the cells, as indicated (left and right panels, respectively). Right lanes refer to wild-type (WT) HEK293 cells. ( D ) Expression and localization of GDE3 and mutant GDE3(H229A), as measured by immunoblotting and confocal microscopy, respectively, at 24 hr after transfection. Actin was used as a loading control. ( E ) uPAR-TM containing the transmembrane domain of EGFR is not released by GDE3, as shown by immunoblotting using anti-uPAR antibody, while uPAR-TM is properly expressed at the plasma membrane (bar, 10 μm).

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Expressing, Super-Resolution Microscopy, Western Blot, Membrane, Plasmid Preparation, Mutagenesis, Confocal Microscopy, Transfection, Control, Clinical Proteomics

Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) (Left) Scheme showing uPAR cleavage in cis or trans. (Right) GDE3-expressing HEK-uPAR cells were mixed with a GDE3-deficient cell population, as indicated. Immunoblot analysis of uPAR in medium and cell lysates indicates that GDE3 acts in cis; mock refers to empty vector-transfected cells. ( B ) GDE3 expression leads to increased GPI-free suPAR. Conditioned medium from HEK-uPAR cells expressing GDE2 or GDE3 was subjected to Triton X-114 phase separation. suPAR in the aqueous ( A ) and detergent ( D ) fractions was analyzed by immunoblotting. ( C ) GPI-anchor with phospholipase cleavage sites indicated; HONO, nitrous acid. ( D ) Representative LC-MS ion chromatograms (m/z 259.02–259.03); inositol 1-phosphate peaks in red. HONO-treated suPAR contains inositol 1-phosphate (n = 3, mean ±SEM; *p<0.05).

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells

doi: 10.7554/eLife.23649

Figure Lengend Snippet: ( A ) Endogenous uPAR expression in MDA-MB-231 versus HEK293 cells, as determined by immunoblot. ( B ) Endogenous GDPD2 expression, as determined by qPCR analysis. ( C ) (left) Cell-surface expression of GDE3-mCherry of MDA-MB-231 cells expressing GDE3, as detected by flow cytometry. (Right) Cell-surface expression of uPAR in control (grey) and GDE3-expressing MDA-MB-231 cells (red), as detected by flow cytometry. ( D ) Confocal (top) and dual-color super-resolution microscopy images (bottom) of MDA-MB-231 cells expressing GDE3-GFP or catalytically dead GDE3(H229A)-GFP. Endogenous uPAR was immunostained in red. Merged images show colocalization of uPAR with GDE3(H229A) but not with wild-type GDE3 and uPAR. Scale bars, 10 μm (confocal) and 1 μm (super-resolution). Co-localization analysis (Mander's coefficient) on peripheral uPAR patches in confocal images was done using ImageJ software (n = 30 cells, three independent experiments). ( E ) Endogenous uPAR staining in control, GDE3-overexpressing and GDE3 knockout MDA-MB-231 cells plated on vitronectin. Two distinct GDE3 knockout clones (KO1 and KO2) were examined, as indicated. Scale bar,10 μm. ( F ) Quantification of basolateral uPAR-containing membrane domains referring to the cells in panel ( E ) (n = 3, mean ±SEM, ****p<0.0001). GDE3 suppresses the vitronectin- and uPAR-dependent phenotype of MDA-MB-231 breast cancer cells.

Article Snippet: To determine the inositol phosphate content of cleaved uPAR, suPAR was immuno-precipitated from HEK293 cell conditioned medium using anti-GFP beads (ChromoTek).

Techniques: Expressing, Western Blot, Flow Cytometry, Control, Super-Resolution Microscopy, Software, Staining, Knock-Out, Clone Assay, Membrane

Journal: Genome Biology

Article Title: Experimental characterization of the human non-sequence-specific nucleic acid interactome

doi: 10.1186/gb-2013-14-7-r81

Figure Lengend Snippet: Validation of preferential affinity . (a) GO molecular function term significance in the various sets of proteins inferred to bind preferentially one or several subtypes of nucleic acids. We observe the clear separation between molecular functions enriched in inferred DNA- and RNA-binding proteins. Color log-scale: red = P < 1E-15, light yellow = P < 0.01, gray = P ≥ 0.01. (b) Examples of affinity preferences of selected NABPs represented by P -values in the statistical analysis (table on left) and western blots in the experimental validation (right). We note the strong agreement between preferred versus non-preferred affinities in the statistics and the blots. (C20orf72 was purified with a Myc tag in HEK293 cells instead of a specific antibody in HepG2 cells.) (c) Methylation specificity usually correlates with CG specificity, but UHRF1 and YB-1 were specific to mCG only in the statistical analysis (see reported P -values in the table on the left). Experimental validation confirmed their specificity (right); AIM2 was used as a DNA-binding non-specific control.

Article Snippet: Myc-tagged C20orf72, AIM2, UHRF1 and YB-1 were overexpressed in HEK293 cells and visualized by immunoblotting using anti-Myc-IRDye800 (Rockland Gilbertsville, PA, USA ).

Techniques: Biomarker Discovery, RNA Binding Assay, Western Blot, Purification, Methylation, Binding Assay, Control

Journal: Veterinary research

Article Title: Inhibition of STING-mediated type I IFN signaling by African swine fever virus DP71L.

doi: 10.1186/s13567-025-01474-3

Figure Lengend Snippet: Figure 3 DP71L interacts with STING. A HEK293T cells were co-transfected with the IFN-β promoter, TK-renilla, plasmids of STING, TBK1, IRF3, and IKKε, and DP71L-Flag for 24 h. The luciferase activity was measured. Results are demonstrated relative to the Renilla luciferase activity. Additionally, 293-Dual hSTING-A162 cells were transfected with DP71L-Flag plasmid, and transfected with poly(dA:dT), 3xFlag-cGAS plasmid or cGAMP for 12 h. The expression of Lucia luciferase, was determined using the Quanti-Luc assay. B The DP71L-GST pull-down assay in HEK293T cells, followed by silver staining and mass spectrometry analysis. C HEK293T cells transfected with DP71L-Strep and STING-GST plasmids. Subsequently, cell lysates were subjected to GST PD, and immunoblotted with anti-Strep and anti-GST antibodies. D PK-15 cells transfected with DP71L-GST and control plasmids, or E 3D4/21 cells stably expressing DP71L-Flag or control plasmids, were stimulated with HSV-GFP (MOI = 1.0). After 24 hpi, cells were harvested and subjected to GST PD or immunoprecipitation with STING antibody, respectively. Immunoblotting was performed with indicated antibodies. F In vitro binding assay was conducted by purified STING-Flag protein incubation with Strep-resin immobilized DP71L-Strep or control-Strep proteins. Bound proteins were eluted and immunoblotted with indicated antibodies. G Confocal microscopy assays were conducted to investigate the co-localization of DP71L and STING proteins in overexpression and endogenous conditions in HeLa and PK-15 cells. After plasmid transfection, cells were stimulated with VACV wild-type virus. The nuclei were stained with DAPI, and the rectangle indicates the co-localization of DP71L and STING proteins at the endogenous level. H HEK293T cells were transfected with GST-tagged DP71L domain constructs and the STING-Strep plasmid. After 36 h, cells were harvested, and WCLs were subjected to a GST PD, followed by immunoblotting with indicated antibodies. Protein sizes are indicated in kDa. The data represent at least three independent experiments with similar results, and the values are expressed as means ± SD for two biological replicates. Student’s t-test: **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: The following cell lines were used: porcine kidney epithelial cells (PK-15) (ATCC CCL-33), HeLa cells (ATCC CCL-2), HEK293T cells (ATCC CRL-11268), Vero cells (ATCC CCL-81), 293-Dual hSTING-A162 cells (InvivoGen, San Diego, CA, USA, 293d-a162), A549 cells (ATCC CCL-185), monkey kidney epithelial cells (MA104) (CRL-2378.1), 3D4/21 cells (ATCC CRL-2843), STING wild-type (STING WT) and STING-knockout (STING KO) HeLa cells.

Techniques: Transfection, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Pull Down Assay, Silver Staining, Mass Spectrometry, Control, Stable Transfection, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Purification, Incubation, Confocal Microscopy, Over Expression, Virus, Staining, Construct