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: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 2. In vitro knockdown of human RHO-T17M expression. (A) Schematic view of construction of 293T stably expressing human RHO protein and transfection of pX601-EFS-SaCas9-U6-sgRNA (SgRNA) plasmid. (B) T7E1 assay indicated that SaCas9/17-Sg1 and SaCas9/17-Sg2 were appeared to cut the mutant sequence specifically, the full-length amplicon was 760 bp, the two truncated amplicons were 510 bp and 250 bp, respectively. (C) The cutting efficacy of two sgRNAs with SaCas9 determined by TA and Sanger sequencing in 293T cells. (D) Rhodopsin expression reduction was determined by WB in RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, comparing to the RHOwt cells with 17-Sg1 and -Sg2 plasmid. (E) Densitometric analysis of immunoblots performed on RHOwt and RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, respectively. The experiment was performed in triplicate and presented as mean ± SEM, the significance was calculated using two-tailed paired t-test, ns = not significant, *p<0.05.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: In Vitro, Knockdown, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Sequencing, Amplification, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 6. Expression of the mutant human RHO allele after gene editing with SaCas9/17-Sg2 in vitro. (A) Schematic view of the different human RHO gene variants created by gene editing. (Top) Map of the pEGFPN1 vector used to overexpress these variants. (Bottom) The description of variants at DNA and protein level. (B) Colocalization of GFP and rhodopsin (4D2, red) in 293T cells transfected with pEGFPN1 vector carrying RHO-WT, RHO-T17M, and four edited RHO-T17M variants, 1 week after transfection. Scale bar = 10 μm. (C) Colocalization of GFP and rhodopsin (4D2, red) in 293T cells transfected with pEGFPN1 vector carrying RHO-5m and four edited RHO-5m variants, 1 week after transfection. Scale bar = 10 μm. (D–F) The number of GFP+ cells and percentage of GFP+ cells expressing rhodopsin per random sight. Nuclei were stained blue by DAPI. Scale bar = 200 μm.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Mutagenesis, In Vitro, Plasmid Preparation, Transfection, Staining

Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 10. Examination of SaCas9/17-Sg2 off-target effects in human gDNA using WGS. Identification of SNVs (A) and indels (C) in 293T cells transfected with 17-Sg2 plasmid at the WGS level. The type of SNVs (B) and indels (D) in 293T cells transfected with 17-Sg2 plasmid and untreated cells at the WGS level.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation

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:

Article Title: Biochemical Characterization of Human Epidermal Retinol Dehydrogenase 2

doi: 10.1016/j.cbi.2008.09.019

Figure Lengend Snippet: HEK293 cells were transfected with either empty vector (A) or RDH-E2 expression vector (B) and incubated with anti-FLAG antibodies. RDH-E2 expression was visualized using FITC-conjugated antibodies. Endoplasmic reticulum of HEK293 cells was visualized by staining with FITC-conjugated concanavalin A (C).

Article Snippet: Analysis of recombinant RDH-E2 expressed in human cells Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium containing 10% horse serum and penicillin/streptomycin at 37 °C with 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Staining

Journal:

Article Title: Biochemical Characterization of Human Epidermal Retinol Dehydrogenase 2

doi: 10.1016/j.cbi.2008.09.019

Figure Lengend Snippet: HEK293 cells transfected with expression vector for RDH-E2 (+) or empty vector (−) were incubated with tritiated androsterone (ADT), dihydrotestosterone (DHT), allopregnanolone (ALLO), dehydroepiandrosterone (DHEA); progesterone (PROG), or corticosterone (CORT).

Article Snippet: Analysis of recombinant RDH-E2 expressed in human cells Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium containing 10% horse serum and penicillin/streptomycin at 37 °C with 5% CO 2 .

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation