Journal: bioRxiv

Article Title: Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

doi: 10.1101/2025.05.10.653258

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of HEK cells transfected with the hGPC4 protein fused to a carboxyterminal HA-tag (hGPC4-CHA). ( B ) Western blot analysis showing exogenous hGPC4 in the total cell lysate and in the membrane fraction used for llama immunization. Note the core protein and all glycanated hGPC4 forms. The N-terminal and C-terminal hGPC4 fragments, generated during or after hGPC4 synthesis and processing are also visible (asterisks) . ( C ) Schematic procedure for the production of phage display Nb libraries. Blood samples were collected at day 28 and day 42 for lymphocyte preparation. ( D ) PCR analysis of phage display library transformants showing that they harbor a vector with an insert of the VHH size. ( E ) Supernatant of individual phage clones analyzed by Western blot. Lane MW: molecular weight marker. Lanes A1 to E2 Nb proteins. The + and – symbols indicated colonies expressing or not the Nb proteins.

Article Snippet: Transfection of the plasmids (around 10 µg for 2 x 10 6 cells in a 10cm tissue culture plate, Corning-Falcon ref. 353003) encoding hGPC4 (Sino Biological, ref HG10090-UT), C-HA-tagged hGPC4 (Sino Biological, ref. HG10090-CY), N-HA-tagged hGPC4 (Sino Biological, ref. HG10090-NY) and mouse Flag-tagged TIGIT (used as irrelevant control molecule; Sino Biological, ref MG50939- NF) was done with Lipofectamine3000 (Invitrogen, ref. L3000015) according to manufacturer’s instruction.

Techniques: Immunofluorescence, Transfection, Western Blot, Membrane, Generated, Plasmid Preparation, Clone Assay, Molecular Weight, Marker, Expressing

Journal: bioRxiv

Article Title: Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

doi: 10.1101/2025.05.10.653258

Figure Lengend Snippet: ( A ) ELISA of the sixteen potential hGPC4 binders selected on recombinant hGPC4-Fc protein. ( B ) Amino acid sequence of the four different Nbs identified. Sequences are numbered according to the Kabat numbering scheme. The complementary determining regions (CDR1, CDR2 and CDR3) were assigned according to the AbM definitions and are shown with a grey background. Based on their amino acid sequences, the Nbs were categorized into four distinct groups: RB1, RB2, RB3, and vRB3. Notably, RB3 and vRB3 are derived from related B-cell clones, differing only by a single amino acid substitution – tyrosine (Y) to asparagine (N) – at the first residue of the framework region 3 (FR3). ( C ) Flow cytometry analysis of binding properties of one representative Nb belonging to the RB1, RB2 and RB3 classes to HeLa cells transfected with hGPC4 expression vectors (red curve), and to HeLa cells transfected with a non-specific plasmid (black curve). Shaded grey areas are the irrelevant Nb (irrNb) signal on the hGPC4 transfected HeLa cells. ( D ) Binding of RB3 to recombinant hGPC4-Fc protein (red curve) and to hIgGs (blue curve) examined by ELISA. RB3 was added at increasing concentrations. Apparent K D were calculated using the GraphPad Prism version 8 software. Data are presented as mean±SEM of n=2. Before pooling, data were normalized by the values of the negative control obtained with a an irrNb. ( E ) Binding of RB1 to HeLa cells expressing either hGPC4 (red curve) or an irrelevant protein (cDNA ctrl; green curve) measured by flow cytometry. Cells were incubated with increasing concentrations of RB1. Apparent K D were calculated using the GraphPad Prism version 8 software. Data are presented as mean±SEM of n=3 biological replicates. Before pooling, data were normalized by the values of the negative control obtained with a an irrNb.

Article Snippet: Transfection of the plasmids (around 10 µg for 2 x 10 6 cells in a 10cm tissue culture plate, Corning-Falcon ref. 353003) encoding hGPC4 (Sino Biological, ref HG10090-UT), C-HA-tagged hGPC4 (Sino Biological, ref. HG10090-CY), N-HA-tagged hGPC4 (Sino Biological, ref. HG10090-NY) and mouse Flag-tagged TIGIT (used as irrelevant control molecule; Sino Biological, ref MG50939- NF) was done with Lipofectamine3000 (Invitrogen, ref. L3000015) according to manufacturer’s instruction.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Sequencing, Derivative Assay, Clone Assay, Residue, Flow Cytometry, Binding Assay, Transfection, Expressing, Plasmid Preparation, Software, Negative Control, Incubation

Journal: bioRxiv

Article Title: Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

doi: 10.1101/2025.05.10.653258

Figure Lengend Snippet: ( A ) Representative results of the GPC4-targeting Nb-screening done by ELISA. Supernatant of individual phage clones (top scheme) was screened by ELISA on plates coated with recombinant hGPC4-Fc (Coating: rGPC4-Fc) or with unrelated Fc tagged recombinant protein as negative control (Coating: rUnrelated-Fc). Values were normalized over those obtained with an irrNb (well C10) and reported as ratio between rGPC4-Fc/rUnrelated-Fc. Positive control: mAb GPC4. Negative controls: Isotype ctrl, irrNb-Fc (Nb-Fc), Irr-Nb, T-, 2 nd Ab. (B) Representative results of the hGPC4-targeting Nb-screening done by Flow cytometry. Nbs were tested for binding properties to HeLA cells expressing hGPC4 (HeLA hGPC4, red curves), HeLA cells transfected with a non-specific plasmid (HeLA cDNA CTRL, green curves) and not transfected cells (HeLA, blue curves). Nbs were added at increasing concentrations (nM). Note that the Nb 3M1_F6 thereafter named as RB1 bind to cells expressing hGPC4. In contrast, 3M1_A2, thereafter named as RB3, does not bind to cells expressing hGPC4. The Nb 3M1_H10, thereafter named as RB2, turned out as not specific hGPC4-binder as it recognizes negative controls. (C) FACS analysis of cell binding on HeLa cells non-transfected (blue curve) or transfected with hGPC4 (red curve) or with an irrelevant cDNA (cDNActrl; green curve). Cells were incubated with increasing concentrations of RB2. n=1 biological replicates. (D) FACS analysis of cell binding on HeLa cells non-transfected (blue curve) or transfected with hGPC4 (red curve) or with an irrelevant cDNA (cDNActrl; green curve). Cells were incubated with increasing concentrations of RB3. n=1 biological replicates. (E) Binding of RB1 to recombinant hGPC4-Fc protein (red curve) and to hIgGs (blue curve) examined by ELISA. RB1 was added at increasing concentrations. Data are presented as mean±SEM of n=2. Before pooling, data were normalized by the values of the negative control obtained with a an irrNb. Note that RB1 binds to recombinant hGPC4-Fc protein with low affinity (Kd apparent >4000nM).

Article Snippet: Transfection of the plasmids (around 10 µg for 2 x 10 6 cells in a 10cm tissue culture plate, Corning-Falcon ref. 353003) encoding hGPC4 (Sino Biological, ref HG10090-UT), C-HA-tagged hGPC4 (Sino Biological, ref. HG10090-CY), N-HA-tagged hGPC4 (Sino Biological, ref. HG10090-NY) and mouse Flag-tagged TIGIT (used as irrelevant control molecule; Sino Biological, ref MG50939- NF) was done with Lipofectamine3000 (Invitrogen, ref. L3000015) according to manufacturer’s instruction.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Recombinant, Negative Control, Positive Control, Flow Cytometry, Binding Assay, Expressing, Transfection, Plasmid Preparation, Incubation

Journal: bioRxiv

Article Title: Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

doi: 10.1101/2025.05.10.653258

Figure Lengend Snippet: ( A ) Schematic representation of the bivalent RB1-FcNb generated by genetically cloning the VHH domain of RB1 in frame with the Fc domain of a human IgG1. ( B ) FACS analysis of cell binding on HeLa cells transfected with hGPC4 (red curve) or with an irrelevant cDNA (cDNActrl; green curve). Cells were incubated with increasing concentrations of RB1-Fc. Data are presented as mean±SEM of n=3 biological replicates. Before pooling, data were normalized by the values of the negative control obtained with a an irrNb. Apparent K D were calculated using the GraphPad Prism version 8 software. ( C ) qRT-PCR analyses of hGPC4 mRNA levels in HeLA, MKN, SNU-449 cancer cells, in hiPSCs and hiPSCs with reduced hGPC4 levels. Levels of all transcripts were normalized to those of the housekeeping gene GAPDH and subsequently to the ΔCt of the A549 cell lines that do not express hGPC4 . Note the different hGPC4 mRNA levels in cell lines. Data are presented as mean±SEM of n=2 biological replicates. One- way ANOVA with Dunnett’s multiple comparison test: *** p<0. 001. ( D ) Flow cytometry analysis of RB1-Fc cell binding on HeLa, MKN, SNU-449 cancer cells, on hiPSCs and hiPSCs with reduced hGPC4 levels. HGPC4 expression levels in different cell lines were evaluated by comparing the fluorescence staining given by RB1-Fc with that of an irrelevant Nb-Fc (irrNb-Fc). Note that despite low level of mRNA in SNU-449, RB1- Fc exhibits comparable binding to that observed in MKN cells, indicating similar surface target availability. ( E ) Flow cytometry analysis of RB1-Fc binding to hiPSCs and hiPSCs with reduced hGPC4 levels applied at 133nM concentrations. Data are presented as mean±SEM of n=3 biological replicates. Before pooling, data were normalized by the values of the negative control obtained with an irrelevant Nb-Fc. ( F ) Immunofluorescence analysis of hGPC4 in different cell lines using RB1-Fc. An irrNb- Fc was used as negative control. RB1-Fc detected hGPC4 in HeLa cells transfected with hGPC4 (HeLa + GPC4) as well as endogenous hGPC4 in cells such as MKN and hiPSCs producing relatively high hGPC4. No staining was detected in HeLa cells or hiPSCs GPC4sh due to their lack or low hGPC4 transcript levels (see panel (C)). A commercially available mGPC4Ab was used as control of GPC4 expression on these cells.

Article Snippet: Transfection of the plasmids (around 10 µg for 2 x 10 6 cells in a 10cm tissue culture plate, Corning-Falcon ref. 353003) encoding hGPC4 (Sino Biological, ref HG10090-UT), C-HA-tagged hGPC4 (Sino Biological, ref. HG10090-CY), N-HA-tagged hGPC4 (Sino Biological, ref. HG10090-NY) and mouse Flag-tagged TIGIT (used as irrelevant control molecule; Sino Biological, ref MG50939- NF) was done with Lipofectamine3000 (Invitrogen, ref. L3000015) according to manufacturer’s instruction.

Techniques: Generated, Cloning, Binding Assay, Transfection, Incubation, Negative Control, Software, Quantitative RT-PCR, Comparison, Flow Cytometry, Expressing, Fluorescence, Staining, Immunofluorescence, Control

Journal: bioRxiv

Article Title: Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

doi: 10.1101/2025.05.10.653258

Figure Lengend Snippet: ( A ) Western blot analysis of cell extracts from non-transfected HEK cells (HEK), transfected with a hGPC4 with an HA tag at the carboxyterminal (HEK + hGPC4-CHA). Proteins were detected by western blot using anti HA antibodies (left) and RB1-Fc (right) followed by HRP-conjugated anti-IgG. Note that the anti HA antibodies succesfully detected the transfected hGPC4-CHA protein. In contrast, RB1-Fc did not reveal any specific band when compared to lysate from non-transfected HEK cells. The observed bands in the RB1-Fc western blot are considered non-specific signal due to FB1-Fc. ( B ) Immunoprecipitation assay with cell extracts from non-transfected HEK cells (HEK), transfected with a hGPC4 with an HA tag at the carboxyterminus (hGPC4-CHA) or a hGPC4 with an HA tag at the amminoterminus(hGPC4-NHA). Protein extracts were prepared using a lysis buffer with the ionic detergent, sodium deoxycholate (non denaturing buffer 2). Immunoprecipitations were performed using RB1-FC, an irrNb- Fc. Immunoprecipitated proteins were detected by western blot using anti HA antibodies. Note that the hGPC4 was immunoprecipitated from native cell lysates incubated with RB1-Fc and not with the irrNb-Fc control. The asterisks in (B) indicate the Glycanated hGPC4 forms. Note that the band of approximately 50 kDa detected in the lanes corresponding to irrNb-Fc represents the irrNb-Fc nanobody, as it is recognized by the anti-HA antibody targeting its HA tag epitope.

Article Snippet: Transfection of the plasmids (around 10 µg for 2 x 10 6 cells in a 10cm tissue culture plate, Corning-Falcon ref. 353003) encoding hGPC4 (Sino Biological, ref HG10090-UT), C-HA-tagged hGPC4 (Sino Biological, ref. HG10090-CY), N-HA-tagged hGPC4 (Sino Biological, ref. HG10090-NY) and mouse Flag-tagged TIGIT (used as irrelevant control molecule; Sino Biological, ref MG50939- NF) was done with Lipofectamine3000 (Invitrogen, ref. L3000015) according to manufacturer’s instruction.

Techniques: Western Blot, Transfection, Immunoprecipitation, Lysis, Incubation, Control

Journal: bioRxiv

Article Title: Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

doi: 10.1101/2025.05.10.653258

Figure Lengend Snippet: ( A-B ) Immunoprecipitation assay with cell extracts from non-transfected HEK cells (HEK) or transfected with a hGPC4 with an HA tag at the carboxyterminal (hGPC4-CHA) or a hGPC4 with an HA tag at the amminoterminal (hGPC4-NHA). Protein extracts were prepared using non denaturing buffer 1 ( A ) or denaturing ( B ) buffers and analysed by SDS-PAGE under reducing conditions. Immunoprecipitations were performed using RB1-Fc, and irrNb-Fc. Immunoprecipitated proteins were detected by western blot using anti HA antibodies. Note that the hGPC4 was immunoprecipitated from native cell lysates incubated with RB1-Fc and not with the irrNb-Fc control (A). The immunoprecipitation of hGPC4 from a denatured cell lysate was found to be inefficient (B). The asterisks in (A) indicate the glycanated hGPC4 forms. Note that the band of approximately 50 kDa detected in the lanes corresponding to irrNb-Fc represents the irrNb-Fc nanobody, as it is recognized by the anti-HA antibody targeting its HA tag epitope.

Article Snippet: Transfection of the plasmids (around 10 µg for 2 x 10 6 cells in a 10cm tissue culture plate, Corning-Falcon ref. 353003) encoding hGPC4 (Sino Biological, ref HG10090-UT), C-HA-tagged hGPC4 (Sino Biological, ref. HG10090-CY), N-HA-tagged hGPC4 (Sino Biological, ref. HG10090-NY) and mouse Flag-tagged TIGIT (used as irrelevant control molecule; Sino Biological, ref MG50939- NF) was done with Lipofectamine3000 (Invitrogen, ref. L3000015) according to manufacturer’s instruction.

Techniques: Immunoprecipitation, Transfection, SDS Page, Western Blot, Incubation, Control

Journal: bioRxiv

Article Title: Functional targeting of Glypican-4 by a conformation-specific single-domain antibody

doi: 10.1101/2025.05.10.653258

Figure Lengend Snippet: ( A ) Viability of hiPSCs in the presence of increasing concentrations of RB1-Fc and irrNb-Fc. Percentage of viable cells was measured in a metabolic activity-based cell viability assay. Data are presented as mean±SEM of n=4 biological replicates. One-Way ANOVA with Tukey’s multiple comparison tests: *** p<0. 001. Note that not significant RB1-Fc toxic effects were observed at concentrations up to 1000nM. ( B ) Schematic representation of the endoderm differentiation protocol of hiPSCs applied to test the RB1-Fc blocking activity on hGPC4. hiPSCs are plated at day 0 and exposed to ACTIVIN A (ActA) and ACTIVIN A + 0.2% FBS (ActA+FBS) at day 1 and day 2 of differentiation, respectively. Endoderm differentiation is analyzed at day 3 by following the number of SOX17 positive cells. ( C ) Representative images of SOX17 positive cells in hiPSCs and hiPSCs with reduced hGPC4 levels (hiPSCs GPC4sh) after 3 days of differentiation showing the increased distribution of endodermal cells in the hiPSCs GPC4sh line. ( D ) Representative images of SOX17 positive cells in hiPSCs exposed to irrNb-Fc control and to RB1-Fc at 50 and 500nM. hiPSCs were treated with the Nbs either from day1 of differentiation or starting from day 0. Note that the increased distribution of SOX17 positive cells in hiPSCs treated with RB1-Fc is comparable to that observed in the hiPSCs GPC4sh line (C). ( E ) Quantitative analysis of SOX17 positive cells in hiPSCs treated with a control (irrNb-Fc) and with RB1-Fc at 500 nM. SOX17-positive cells were defined as those whose mean fluorescence intensity was above the 95 th percentile of the mean SOX17 intensity in negative cells at the same time point. At least 10 randomly selected frames of view were taken per condition, covering >2.23 mm² in total. Data are presented as mean±SEM of n=3 biological replicates. One-Way ANOVA with Tukey’s multiple comparison tests: ** p<0. 01. Note a ∼10 % increase of SOX17 positive cells in differentiating hiPSCs incubated with RB1-Fc in comparison to hiPSCs incubated with an irrelevant Nb. Also, hiPSCs exposed to RB1-Fc at day 0 have a higher trend for generating SOX17 positive cells in comparison to cells treated with RB1-Fc starting from day1.

Article Snippet: Transfection of the plasmids (around 10 µg for 2 x 10 6 cells in a 10cm tissue culture plate, Corning-Falcon ref. 353003) encoding hGPC4 (Sino Biological, ref HG10090-UT), C-HA-tagged hGPC4 (Sino Biological, ref. HG10090-CY), N-HA-tagged hGPC4 (Sino Biological, ref. HG10090-NY) and mouse Flag-tagged TIGIT (used as irrelevant control molecule; Sino Biological, ref MG50939- NF) was done with Lipofectamine3000 (Invitrogen, ref. L3000015) according to manufacturer’s instruction.

Techniques: Activity Assay, Viability Assay, Comparison, Blocking Assay, Control, Fluorescence, Incubation

Journal: Glycobiology

Article Title: LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans.

doi: 10.1093/glycob/cww075

Figure Lengend Snippet: Fig. 2. Overexpression of GPC4 enhances the IIH6 immunoreactivity of the LARGE2-expressing DG−/−cells. (A) Schematic representation of the myc-tagged GPC4 (Myc-GPC4) construct. The suggested proteolytic cleavage site and potential GAG attachment sites are indicated by an arrow and vertical lines, respect- ively. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. The DG−/−cells stably expressing LARGE2 were transfected with Myc-GPC4. (B) Clones stably expressing myc (#4, #14 and #22) were analyzed by flow cytometry for cell-surface staining with IIH6 or anti-myc antibody. (C) Lysates of these cells enriched for Myc-GPC4 by DEAE-enrichment were immunoblotted with IIH6 or anti-myc antibody. Note that the enhanced intensity of the IIH6 staining is correlated with that of anti-myc staining. (D) Representative immunofluorescence micrograph of the DG−/−cells stably co-expressing LARGE2 and Myc-GPC4 (clone #4). The cell surface was stained with IIH6 and anti-myc antibody in the absence of permeabilization. Scale bar, 50 μm. The top image shows IIH6 immunofluorescence, the middle image shows myc immunofluorescence, and the bottom image shows IIH6 (red) merged with Myc (green) and DAPI (blue). All myc staining coloca- lizes with IIH6 as no ‘green only’ fluorescence is seen. This figure is available in black and white in print and in color at Glycobiology online.

Article Snippet: First, a fragment containing the SacI site and a c-myc sequence with an internal BamHI site were amplified by PCR, using primers 5′-AGAGCTCGAACAAAAGCTGATTTCTG AAGAAGACCTCAAGTCGAAAAGTTGCTC-3′ and 5′-CACAG CCTTGGAAAACCTTC-3′ and the template mouse GPC4 cDNA (BC006622, OriGene, Rockville, MD).

Techniques: Over Expression, Expressing, Construct, Sequencing, Stable Transfection, Transfection, Clone Assay, Cytometry, Staining

Journal: Glycobiology

Article Title: LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans.

doi: 10.1093/glycob/cww075

Figure Lengend Snippet: Fig. 3. LARGE2 can modify GPC4 with the laminin-binding glycan. (A) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleavage site. Vertical lines, potential GAG attachment sites. ss, signal sequence. GPI ss, GPI-anchoring signal sequence. (B) LARGE2 can modify GPC4Fc in CHO cells. Immunoblotting of the Fc fusion proteins transiently expressed in, and purified from, the media of CHO cells with or without stable expression of LARGE1 or LARGE2. (C) Immunoblotting or laminin overlay (OL) of GPC4Fc purified from serum-free CHO culture with or without stable expression of LARGE2. Treatment with neither heparinase (D) nor aqHF (E) removed the functional modification from GPC4Fc.

Article Snippet: First, a fragment containing the SacI site and a c-myc sequence with an internal BamHI site were amplified by PCR, using primers 5′-AGAGCTCGAACAAAAGCTGATTTCTG AAGAAGACCTCAAGTCGAAAAGTTGCTC-3′ and 5′-CACAG CCTTGGAAAACCTTC-3′ and the template mouse GPC4 cDNA (BC006622, OriGene, Rockville, MD).

Techniques: Binding Assay, Glycoproteomics, Construct, Sequencing, Western Blot, Expressing, Functional Assay

Journal: Glycobiology

Article Title: LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans.

doi: 10.1093/glycob/cww075

Figure Lengend Snippet: Fig. 5. LARGE2 can modify not only GPC4 but also other PGs. (A) Schematic representation of Fc-fusion constructs. Dotted arrow, suggested proteolytic cleav- age site. Vertical lines, potential GAG attachment sites. (B) LARGE2 can modify PGs. Immunoblotting of Fc fusion proteins transiently expressed in the WT CHO cells with or without stable expression of LARGE2. (C) Immunoblotting of Fc-fusion proteins transiently expressed in HS- or GAG-deficient CHO mutant cells that stably express LARGE2. In the GAG-deficient cells, LARGE2 could not modify GPC4Fc or BGNFc.

Article Snippet: First, a fragment containing the SacI site and a c-myc sequence with an internal BamHI site were amplified by PCR, using primers 5′-AGAGCTCGAACAAAAGCTGATTTCTG AAGAAGACCTCAAGTCGAAAAGTTGCTC-3′ and 5′-CACAG CCTTGGAAAACCTTC-3′ and the template mouse GPC4 cDNA (BC006622, OriGene, Rockville, MD).

Techniques: Construct, Western Blot, Expressing, Mutagenesis, Stable Transfection

Journal: Scientific Reports

Article Title: The effects of Rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach

doi: 10.1038/srep09167

Figure Lengend Snippet: Information of primary antibody used in this study

Article Snippet: Glypican-4 (MAFB4044) , Creative Biomart (CAB-244MH) , Fix and Perm (Medium A) 15 minutes, room temp. , 10 μg/mL.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes

doi: 10.3390/ijms25073945

Figure Lengend Snippet: GPC4 displays a wide range of expression differences between normal and tumor tissues across TCGA cancer types. Volcano plot depicts median log2 fold change ( x -axis and color scale) of GPC4 expression between tumor and normal subjects across 24 TCGA cancer types. Adjusted Wilcoxon rank sum test p values (−log10) are shown on the y -axis. Horizontal dashed line denotes the 0.05 p value threshold.

Article Snippet: Transfection was performed using either a human GPC4 overexpression plasmid containing the C-GFPSpark tag (overexpression GPC4 ) (cat# HG10090-ACG; Sino Biological, Düsseldorf, Germany) or a non-specific control plasmid with the C-GFPSpark tag and not targeting any known gene (overexpression control) (cat#CV026; Sino Biological, Düsseldorf, Germany).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes

doi: 10.3390/ijms25073945

Figure Lengend Snippet: Association between GPC4 expression and cancer prognosis. ( A ) Hazard ratio of GPC4 expression levels on overall survival across TCGA cancer types. Cox proportional hazard p values are denoted next to each error bar. Error bars represent 95% confidence intervals. ( B ) Kaplan-Meier curves of the survival probability of GPC4 expression strata in indicated TCGA projects in ( A ) where expression levels are associated with significant changes in cancer outcome. List of TCGA cancer abbreviations has been provided in .

Article Snippet: Transfection was performed using either a human GPC4 overexpression plasmid containing the C-GFPSpark tag (overexpression GPC4 ) (cat# HG10090-ACG; Sino Biological, Düsseldorf, Germany) or a non-specific control plasmid with the C-GFPSpark tag and not targeting any known gene (overexpression control) (cat#CV026; Sino Biological, Düsseldorf, Germany).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes

doi: 10.3390/ijms25073945

Figure Lengend Snippet: Suppression of GPC4 expression attenuates proliferation of glioblastoma and augments proliferation of lung adenocarcinoma cells, whereas overexpression of GPC4 augments proliferation of glioblastoma and attenuates proliferation of lung adenocarcinoma cells. ( A , B ) Depletion of GPC4 expression by CRISPR/Cas 9 and ( C , D ) overexpression of GPC4 by overexpression vector, in SNB-75, SF-295 and HOP-92 cells (as indicated in the images). ( A ) Depletion of endogenous GPC4 expression by CRISPR/Cas9, as measured by immunofluorescence microscopy (60× magnifications). Cells were transfected with a control double nickase plasmid (not targeting any known gene; CRISPR Control) or a CRISPR/Cas9 double nickase plasmid construct targeting GPC4 (CRISPR/Cas9 GPC4). ( C ) Overexpression of GPC4 by overexpression vector, as measured by immunofluorescence microscopy (60× magnifications). Cells were transfected with a pCMV3-C-GFPSpark negative control vector not targeting any known gene (Overexpression Control) or a pCMV3-C-GFPSpark GPC4 overexpression vector (Overexpression GPC4). Both CRISPR and overexpression constructs as well as control vectors encoded a GFP reporter to visualize successful transfection. After fixation with acetone the cells were stained with GPC4 antibody followed by Alexa Fluor 594-tagged goat anti-rabbit IgG. Expression of double nickase plasmids (GFP) and silencing or overexpression of GPC4 (Alexa Fluor 594) was monitored by fluorescence microscopy. To visualize the cells, their nuclei were counterstained with DAPI (blue). The same exposure time was used in all experiments. Bar, 20 μm. Insets in ( A , C ): The intensity of GPC4 signal was determined in 4 identical low-magnification immunofluorescence images (20× magnification) and expressed in diagrams as mean intensity values ± SE. The level of immuno-reactive GPC4 was significantly suppressed in the CRISPR/Cas9 GPC4 cells and increased in GPC4-overexpressed cells as compared to controls (Student’s t -test, two-tailed unequal variances, n = 4, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001). ( B , D ) Effect of disruption of GPC4 expression on proliferation of SNB-75, SF-295, and HOP-92 cells. SNB-75, SF-295, and HOP-92 cells were transfected with either ( B ) CRISPR control or CRISPR/Cas9 GPC4 vector or ( D ) overexpression control vector or GPC4 overexpression vector as indicated in the images. After 3 days of proliferation, the cell densities were determined. Controls cells were left untreated cells containing only culture medium. The relative cell numbers were calculated as % of untreated cells. The graphs show results for double experiments ( n = 6 in each experiment). Means ± SE are shown for each data point. The proliferation rate of SNB-75 and SF-295 cells was significantly decreased and the proliferation rate of HOP-92 cells was significantly increased in the CRISPR/Cas9 GPC4 transfected cells in comparison with the control cells (Student’s t -test, two-tailed unequal variances, n = 6). In contrast, the proliferation of SNB-75 cells exhibited a significant increase, while the proliferation of HOP-92 cells showed a significant decrease when transfected with the GPC4 overexpression vector in comparison with the control vector (Student’s t -test, two-tailed unequal variances, n = 6). p ≤ 0.05 was considered as statistically significant. p values are indicated as following: ns (not significant) p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Transfection was performed using either a human GPC4 overexpression plasmid containing the C-GFPSpark tag (overexpression GPC4 ) (cat# HG10090-ACG; Sino Biological, Düsseldorf, Germany) or a non-specific control plasmid with the C-GFPSpark tag and not targeting any known gene (overexpression control) (cat#CV026; Sino Biological, Düsseldorf, Germany).

Techniques: Expressing, Over Expression, CRISPR, Plasmid Preparation, Immunofluorescence, Microscopy, Transfection, Construct, Negative Control, Staining, Fluorescence, Two Tailed Test, Disruption, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes

doi: 10.3390/ijms25073945

Figure Lengend Snippet: Overexpression of GPC4 restores previously high proliferative rate following GPC4 knockdown in gliobalstoma cells. Graphs show mean staining intensities measured at A595 nm for four different treatments (CRISPR Control, CRISPR/Cas9 GPC4, CRISPR/Cas9 GPC4 followed by overexpression control vector and CRISPR/Cas9 GPC4 followed by GPC4 overexpression vector; n = 6 in each experiment). Means ± SE are shown for each experiment. The proliferative rate was significantly decreased for both SNB-75 and SF-295 cells treated with CRISPR/Cas9 GPC4 or CRISPR/Cas9 GPC4 followed by the overexpression control vector compared to cells treated with CRISPR/Cas9 GPC4 followed by the GPC4 overexpression vector (Student’s t -test, two-tailed unequal variances, n = 6). Differences in staining intensities when comparing cells treated with CRISPR control or CRISPR/Cas9 GPC4 followed by GPC4 overexpression vector were non-significant for both cell lines (Student’s t -test, two-tailed unequal variances, n = 6). p ≤ 0.05 was considered as statistically significant. p values are indicated as follows: ns (not significant) p > 0.05, ** p ≤ 0.01, **** p ≤ 0.0001, ***** p ≤ 0.00001 and ******** p ≤ 0.00000001.

Article Snippet: Transfection was performed using either a human GPC4 overexpression plasmid containing the C-GFPSpark tag (overexpression GPC4 ) (cat# HG10090-ACG; Sino Biological, Düsseldorf, Germany) or a non-specific control plasmid with the C-GFPSpark tag and not targeting any known gene (overexpression control) (cat#CV026; Sino Biological, Düsseldorf, Germany).

Techniques: Over Expression, Staining, CRISPR, Plasmid Preparation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes

doi: 10.3390/ijms25073945

Figure Lengend Snippet: Pathway analyses of 352 genes that exhibited differential expression patterns in at least 10 cancer types between patients with high and low GPC4 expression levels revealed the involvement of mechanisms associated with cancer cell proliferation, migration, and immunological aspects of cancer. ( A ) Network reconstruction of the predicted molecular relationships as inferred from gene expression changes between GPC4 -high and GPC4 -low cancer patients (orange: predicted activation; blue: predicted inhibition). ( B ) Dotplot presentation illustrating the top 10 canonical pathways enriched by enrichment ratio ( x -axis). Color-coded predicted activity z -score where z -score > 0 indicates activation and z -score < 0 indicates inhibition. Pathways with negligible or no prediction (near-zero or no or prediction) are shown in gray. The size of the dots corresponds to the Benjamini-Hochberg adjusted p value (−log10).

Article Snippet: Transfection was performed using either a human GPC4 overexpression plasmid containing the C-GFPSpark tag (overexpression GPC4 ) (cat# HG10090-ACG; Sino Biological, Düsseldorf, Germany) or a non-specific control plasmid with the C-GFPSpark tag and not targeting any known gene (overexpression control) (cat#CV026; Sino Biological, Düsseldorf, Germany).

Techniques: Expressing, Migration, Activation Assay, Inhibition, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Dichotomous Effects of Glypican-4 on Cancer Progression and Its Crosstalk with Oncogenes

doi: 10.3390/ijms25073945

Figure Lengend Snippet: Discordant gene expression profiles explain the differential effects of GPC4 upregulation between lung adenocarcinoma and glioblastoma. ( A ) Scatterplot depicting the log2 fold changes of genes found differentially expressed both in TCGA-LUAD ( y -axis) and TCGA-GBM ( x -axis) subjects between GPC4 -high and GPC4 -low cohorts. Genes that are upregulated in glioblastoma but downregulated in lung adenocarcinoma are highlighted in red. ( B ) Top 10 significant terms from pathway enrichment analysis results from 3 databases of the 12 differentially expressed genes with discordant profiles between TCGA-LUAD and TCGA-GBM. Combined enrichment score is plotted on x -axis. Enrichment log-odds over background is encoded as the bullet color while the bullet size encodes the enrichment adjusted p value (−log10).

Article Snippet: Transfection was performed using either a human GPC4 overexpression plasmid containing the C-GFPSpark tag (overexpression GPC4 ) (cat# HG10090-ACG; Sino Biological, Düsseldorf, Germany) or a non-specific control plasmid with the C-GFPSpark tag and not targeting any known gene (overexpression control) (cat#CV026; Sino Biological, Düsseldorf, Germany).

Techniques: Expressing

Journal: Neuron

Article Title: An input-specific orphan receptor GPR158-HSPG interaction organizes hippocampal mossy fiber-CA3 synapses

doi: 10.1016/j.neuron.2018.08.038

Figure Lengend Snippet: (A) Proteomic workflow for the identification of GPC4-interacting proteins.

Article Snippet: GPC4-Fc, GPC4ΔGAG-Fc and GPR158-Fc proteins were produced by transient transfection of HEK293T cells using PEI (Polysciences).

Techniques: