Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a 60 ng of RNA from PCa patients was utilized to assess RNA sequencing data for gene expression ( n = 20) comprised of patients with de novo metastatic (clinical stage M1, n = 10) as well as presumed localized (clinical stage P0, n = 10) tumors. Heatmap shows 25 significantly (FDR ≤ 0.05) differentially expressed genes (ranked by log2(fold change) value) across two comparisons (primary tumors vs. metastatic tumors). The log2(fold change) and -log10(FDR) were shown on the left side of the heatmap. b – d Four prostate cancer cell lines were incubated in 1640 medium with 10% FBS, and total protein ( b , c ) or RNA ( d ) was extracted. Western blot analysis was performed to analyze GIPC2 protein expression in each cell line, whereas real-time RT-PCR was performed to analyze GIPC2 mRNA expression. The protein- and mRNA-expression levels were normalized to those of ACTB. e GIPC2 mRNA expression in primary prostate cancer tissues ( n = 36), metastatic prostate cancer tissues ( n = 17), and normal adjacent tissues ( n = 36), these tissues all derived from 53 patients. Relative to normal adjacent tissues, GIPC2 trended toward decreased expression in primary prostate cancer tissues and displayed significantly increased expression in metastatic prostate cancer tissues (*** p < 0.0001, two-sided Mann–Whitney test). f Evaluation of GIPC2 staining in prostate cancer tissues. GIPC2 IHC staining was performed with primary prostate cancer and metastatic prostate cancer tissues, as well as normal adjacent tissues, and analyzed statistically. g Positive IHC-staining areas in each group were analyzed using Image J software. The data shown represent the mean ± SD of three independent experiments. Scale bar, 100 µm. ** p < 0.01. h Overall survival analysis versus GIPC2 expression.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: RNA Sequencing, Gene Expression, Incubation, Western Blot, Expressing, Quantitative RT-PCR, Derivative Assay, MANN-WHITNEY, Staining, Immunohistochemistry, Software

Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a Schematic representation of the locations of CpG islands. The red bar shows the CpG sites. The corresponding numbers indicate the genomic locations designated in the UCSC database (hg38). b The promoter-methylation ratios of GIPC2 in prostate cancer samples were detected by EpiTYPER methylation analysis. The methylation ratios were significantly higher in normal adjacent tissues than in prostate cancer tumors. c Correlation between methylation of GIPC2-promoter CpG islands and their expression levels in all samples ( n = 89). d The promoter-methylation ratios of GIPC2 were determined by MSP in four cell lines treated with or without DAC. Unmethylated (Unmeth) and methylated (Meth) PCR products were detected. After DAC treatment, GIPC2 mRNA- and protein-expression levels were detected using RT-PCR ( e ) and western blotting ( f , g ). The error bars represent the mean ± SD. h Effect of DAC expression on the methylation status of the GIPC2 promoter. DNA from control- or DAC-treated RWPE-1 cells was collected at the indicated time points, cloned, and sequenced to detect CpG-island methylation at the GIPC2 promoter. A summary of bisulfite-treated gDNA-sequencing results from RWPE-1 cells treated with DAC for increasing times is shown, where the amplified region contained 14 CpG sites (represented by circles located along the region) were analyzed by DNA sequencing. The black and white circles represent methylated and unmethylated CpG dinucleotides, respectively. Each line represents the DNA sequence of a random clone, for which the black and white circles represent unmethylated and methylated CpG sites in these regions, respectively. i , j RWPE-1 cells were treated with DAC, and GIPC2 protein expression was detected at 0, 24, 48, or 72 h.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Clone Assay, Sequencing, Amplification, DNA Sequencing

Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a – d EdU staining and ( e , f ) CCK8 assays were performed to evaluate the effect of GIPC2 on cell proliferation. RWPE-1 and C4-2 cells were transfected with pcDNA3.1-GIPC2 or siRNA of GIPC2 (siGIPC2-1/siGIPC2-2), and cell proliferation was detected by EdU staining and a cell-viability kit at different time points. Apoptosis was determined using a Cell Death Detection Kit ( g , h ) and FCM ( i , j ). GIPC2 overexpression in RWPE-1 cells and GIPC2 knockdown in C4-2 cells. All experiments were performed at least thrice and the data shown represent the mean ± SD. * p < 0.05, ** p < 0.01, two-sided paired t test.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: Staining, Transfection, Over Expression, Knockdown

Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a , b Cell-migration abilities were measured by performing wound-healing assays. Transfecting RWPE-1 and C4-2 cells with pcDNA-GIPC2 or siGIPC2-1/siGIPC2-2 attenuated the pro-migration and pro-invasion effects of GIPC2. After scratching a wound and removing the floating cells, prostate cancer cells (without treatment or control-treated) were tested in wound-scratch assays for 0–3 days. c – j Cell invasion was analyzed by performing transwell assays and the microfluidic platform. c , d Prostate cancer cells treated as described above were applied to transwell chambers coated with Matrigel and incubated for 24 h. e – h Representative fluorescent images of RWPE-1 and C4-2 cell adhesion (green: Calcein-AM). In these images, the endothelial barrier region was treated with 100 ng/ml EGF. Adhesion selectivity to EGF-stimulated was comparable for RWPE-1 and C4-2 cells, including GIPC2-overexpression RWPE-1 cells and GIPC2-knockdown C4-2 cells. i , j The microfluidic model consists of two independent microchannels, where C4-2 cells and 20% FBS were seeded. Between the two channels, 3D Matrigel was seeded to mimic the ECM. Invading cells were labeled with Calcein-AM (green). The data shown represent the mean ± SD. * p < 0.05; ** p < 0.01. k Schematic representation of a mouse model of prostate cancer metastasis. l , m C4-2-luc Control or shRNA-GIPC2-1 and DU145-luc Control or shRNA-GIPC2-1 were suspended in 15 μL sterile PBS and used for intracardiac injections into male BALB/c nu/nu mice ( n = 8 of each group, 32 mice in all). Bioluminescent images of metastatic tumors were monitored. Metastasis rates were determined according to the Kaplan–Meier method. * p < 0.05 between two groups. n – p The metastatic burden of different organs was quantified by monitored using an IVIS Lumina II ( n = 3 of each group). Colored scale bars represent low (purple) to high (red) tumor burdens. q Paraffin sections of organs were stained with hematoxylin and eosin (H&E) and the tumor tissue is indicated by an arrow. r Upregulated and downregulated genes in GIPC2-overexpression RWPE-1 cells were imported into IPA. Through core analysis, the top associated networks and functions were found, and the first and the only meaningful pathway is shown. Red shading represents upregulated genes and green shading represents downregulated genes in the microarray.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: Migration, Control, Incubation, Over Expression, Knockdown, Labeling, shRNA, Sterility, Staining, Microarray

Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a , b RWPE-1 and C4-2 cells were transfected with pcDNA-GIPC2 or siGIPC2-1/siGIPC2-2 for 48 h. Then, the protein-expression levels of GIPC2, p-GSK-3β, total GSK-3β, activated β-catenin, and total β-catenin were analyzed by western blotting, with ACTB serving as a loading control. c , d Graphical display of target protein-expression levels normalized to ACTB expression. e , f Treating RWPE-1 cells with 0.25 μg DKK1 (an inhibitor of the Wnt–β-catenin-signaling pathway), with or without GIPC2 overexpression. Total proteins were harvested at the indicated time points for western blot analysis. g , h C4-2 cells were treated with 5 mM LiCl (an activator of the Wnt–β-catenin-signaling pathway) for 24 h, following GIPC2 downregulation. Protein-expression levels of GIPC2, p-GSK-3β, total GSK-3β, activated β-catenin, and total β-catenin were analyzed by western blotting, with ACTB serving as a loading control. i , j In tumor tissue homogenates from mouse xenografts, protein expression levels of GIPC2, p-GSK-3β, and activated β-catenin were measured to verify the effects of GIPC2 knockdown. Western blot-band intensities were measured using Image J software. Normalization was done by dividing the target signal by the ACTB signal. P values were determined by Student’s t test. The results are presented as the mean ± SD. * p < 0.05, n = 3.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: Transfection, Expressing, Western Blot, Control, Over Expression, Knockdown, Software

Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a , b Exogenous GIPC2 associated with exogenous Fzd7. Total lysates from C4-2 cells expressing GIPC2-HA and/or Fzd7-Myc were immunoprecipitated with antibodies against Myc ( a ) or the HA tag ( b ), followed by western blotting using the indicated antibodies. c Endogenous GIPC2 interacted with endogenous Fzd7. C4-2 cell lysates were immunoprecipitated with an anti-Fzd7 antibody, followed by western blotting using an anti-GIPC2 antibody. d C4-2 cells were treated with or without DAC. Endogenous GIPC2 from C4-2 cells was analyzed by western blotting with an anti-Fzd7 antibody or immunoprecipitated with an anti-GIPC2 antibody. e Positive FRET images. C4-2 cells, co-expressing pmCherry-Fzd7 and three different GFP–GIPC2 domains, were analyzed by confocal microscopy. Positive FRET image of C4-2 cells co-transfected with pairs of GFP-C1 and Red-N1 fusion crystallins. f Negative FRET images. Images were acquired before and after photobleaching. A nonbleached region and a corresponding bleached region (shown by an arrow) were used for the data analysis, compared with an unbleached cellular region as a negative control. g C4-2 cells co-transfected with pAcGFP-GIPC2-PDZ (green) and pmCherry-Fzd7 (red). h C4-2 cells co-transfected with pAcGFP-GIPC2-GH1 and pmCherry-Fzd7. i The images show C4-2 cells co-transfected with pAcGFP-GIPC2-GH2 and pmCherry-Fzd7. j FRET efficiencies (percentages) for the interaction between two crystallin partners were calculated, and the FRET intensity was analyzed statistically. The FRET intensity expressed as a percent represents the fraction of interacting donor molecules. The results suggested that a significant interaction occurred between GIPC2-PDZ and Red-Fzd7. k Domain architecture of GIPC2. The GIPC2 protein consists of a GH1 domain (red), a PDZ domain (blue), and a GH2 domain (green). The amino acid position is shown on the domain architecture. The GH1 domain in the N-terminal region, the PDZ domain in the middle region, and the GH2 domain in the C-terminal region are well conserved among GIPC family members.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: Expressing, Immunoprecipitation, Western Blot, Confocal Microscopy, Transfection, Negative Control

Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a Luciferase-expressing C4-2 cells (2 × 10 5 ) were suspended in 15 μL of sterile PBS and used for intracardiac injections of male BALB/c nu/nu mice with different GIPC2 isoforms (wild-type GIPC2, GIPC2-δGH1, GIPC2-δGH2, and GIPC2-δPDZ, n = 8 of each group). Bioluminescent images of metastatic tumors were monitored. Each picture includes four different time points: 7, 14, 21, and 28 days. The colored scale bars represent low (purple) to high (red) tumor burdens. b The metastasis rate was determined using the Kaplan–Meier method. * p < 0.05 between GIPC2-δPDZ group and three other groups. c Western blotting showed GIPC2, Fzd7, p-GSK-3β, and activated β-catenin expression in C4-2 cells transfected with OE-GIPC2 (GIPC2 overexpression) or si-Fzd7. d Graphical display of target protein-expression levels normalized to ACTB expression. e Cell-migration abilities were measured by performing wound-healing assays. C4-2 cells were transfected with pcDNA-GIPC2 (with or without si-Fzd7) to evaluate the pro-migration and pro-invasion effects of the GIPC2–Fzd7 axis. After scratching a wound and removing the floating cells, prostate cancer cells (untreated or control-treated) were used in wound-scratch assays for 0, 1, or 3 days. f Prostate cancer cells treated as described above were applied to transwell chambers coated with Matrigel and incubated for 24 h. g The microfluidic model consists of two independent microchannels, where tumor cells and 20% FBS were seeded. Between the two channels, 3D Matrigel was seeded to mimic the ECM. Invading cells was observed under a light microscope (200×). The data shown represent the mean ± SD. * p < 0.05; ** p < 0.01.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: Luciferase, Expressing, Sterility, Western Blot, Transfection, Over Expression, Migration, Control, Incubation, Light Microscopy

Journal: Oncogene

Article Title: GIPC2 interacts with Fzd7 to promote prostate cancer metastasis by activating WNT signaling

doi: 10.1038/s41388-022-02255-4

Figure Lengend Snippet: a , b Exosomes were isolated from the supernatants of prostate cancer cell lines. Exosome-marker proteins (including CD9 and TSG101) were analyzed by western blotting. GIPC2 was also observed in supernatants and exosomes from prostate cancer cell lines. c , d The characteristics of exosomes from primary prostate cancer samples and metastatic prostate cancer patients were investigated by western blotting, based on GIPC2 expression and the exosome-specific expression of CD9 and TSG101. e , f Cell-migration abilities were measured by performing wound-healing assays. Treating RWPE-1 cells with exosomes isolated from RWPE-1, GIPC2-overexpression or empty vector-overexpression tumor cells (C4-2 and DU145) to investigate pro-migration and pro-invasion effects. After scratching a wound and removing the floating cells, RWPE-1 cells (untreated or control-treated) were used in wound-scratch assays for 0–3 days. g , h Cell invasion was analyzed in transwell assays and with the microfluidic platform. C4-2 treated as described above were applied to transwell chambers coated with Matrigel and incubated for 24 h. The microfluidic model consists of two independent microchannels, where tumor cells and 20% FBS were seeded. Between the two channels, 3D Matrigel was seeded to mimic the ECM. Invading cells were labeled with Calcein-AM (green). The data shown represent the mean ± SD. * p < 0.05; ** p < 0.01. i ROC-curve analysis for GIPC2 showed higher AUC values for distinguishing between primary or metastatic prostate cancer (** p < 0.01, *** p < 0.0001, two-sided Mann–Whitney test). j Schematic representation of the mechanism whereby GIPC2 promotes tumorigenesis in prostate cancer. The GIPC2–PDZ–Fzd7 pathway in prostate cancer is shown.

Article Snippet: Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution.

Techniques: Isolation, Marker, Western Blot, Expressing, Migration, Over Expression, Plasmid Preparation, Control, Incubation, Labeling, MANN-WHITNEY

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: ( A ) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. Genotypes indicated on top of each image in yellow font. Angiogenesis deficits are indicated as follows: white asterisks (DLAV gaps), magenta asterisks (truncated Se), white greater-than sign (thin Se). Maternal-zygotic (MZ) removal of gipc activity is denoted by the designation ‘MZ’ in superscript. In the WT image (top left), the vessels are designated with the white font as follows: DLAV (Dorsal Longitudinal Anastomotic Vessel), Se (Segmental Vessel), DA (Dorsal Aorta), and PCV (Posterior Cardinal Vein). ( B ) Bar graph. Percentage of Se in 32 hpf embryos of the indicated genotypes belonging to each of the following four phenotypic classes. Truncated: severe (includes missing Se), medium (yellow), and weak (gray). Non-truncated: complete (black). Significance values were calculated using a two-sided Fisher Exact test and significant differences (p<0.0033) assigned using a Bonferroni-type adjustment for 15 pairwise genotype comparisons (0.05/15 = 0.0033). Brackets and asterisks indicate pairs of genotypes with significantly different distributions of these four phenotypic classes. Quantifications. We scored Se angiogenesis in embryos of the following six genotypes: WT (138 Se, 12 embryos; an average of 11.5 Se/embryo), gipc1 skt1 (130 Se, 11 embryos; an average of 11.8 Se/embryo) , gipc1 skt1(MZ) (380 Se, 33 embryos; an average of 11.5 Se/embryo) , gipc2 skt3/skt4 (130 Se, 11 embryos; an average of 11.8 Se/embryo) , gipc1 skt1 ; gipc2 skt3/skt4 (152 Se, 13 embryos; an average of 11.6 Se/embryo), and gipc1 skt1(MZ) ; gipc2 skt3/skt4 (220 Se, 19 embryos; an average of 11.5 Se/embryo). For additional data, graphs, and statistical comparisons related to this figure, see , and . Please note that given the use of different scales for scoring angiogenesis deficits, it is unfeasible to compare the quantifications in and directly.

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Activity Assay

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: ( A–D ) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. Morpholino injection (un-injected or injected with plxnd1 morpholino) indicated on top, genotypes (WT or gipc1 skt1(MZ) ; gipc2 skt4(MZ) ) indicated on the left. The un-injected WT picture ( A ) shows the names of the major vessels in white font: DLAV (Dorsal Longitudinal Anastomotic Vessel), Se (Segmental Vessel), DA (Dorsal Aorta), and PCV (Posterior Cardinal Vein). Vascular defects highlighted as follows: truncated or missing Se (magenta asterisk), thin Se (white greater/less-than signs), DLAV gaps (white asterisk). Quantifications. The following number of embryos were analyzed: WT (four embryos), WT injected with plxnd1 morpholino (four embryos; 4/4 showed a vascular phenotype similar to that of plxnd1 fov01b nulls), gipc1 skt1(MZ) ; gipc2 skt4(MZ) (12 embryos; 7/12 showed angiogenesis deficits), and gipc1 skt1(MZ) ; gipc2 skt4(MZ) injected with plxnd1 morpholino (11 embryos; 11/11 showed a vascular phenotype similar to that of plxnd1 fov01b nulls).

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Injection

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: ( A–F ). Representative fluorescent images of HUVEC morphology in cell collapse experiments under the following conditions. No ligand ( A–C ; top) or 45 min stimulation with 10 nM of SEMA3E ( D–F ; bottom). In each picture, the square area marked by yellow dotted sides is shown at twice the magnification at the bottom left corner and delimited by white sides. shRNA treatments as follows. Non-targeting, control ( A, D ), GIPC ( GIPC1, GIPC2, and GIPC3 ; B, E ), and PLXND1 ( C, F ). Scale bars (white horizontal lines), 100 μm. ( A–C ) Without ligand stimulation cells are uncollapsed regardless of the knockdown condition. ( D, E ) Cell collapse under ligand stimulation. Cells treated with non-targeting, control shRNA collapse ( D ). GIPC knockdown cells hypercollapse ( E ). SEMA3E-induced collapse is PLXND1-dependent. PLXND1 knockdown abrogates the morphological response ( F ). Cell collapse data collected from three independent experiments. This figure is related to .

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: shRNA, Control, Knockdown

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet: Western blots for GIPC1, GIPC2, PLXND1, and GAPDH (loading control) from TCLs of cells infected with the indicated shRNA lentiviral particles. Note the effective decrease of GIPC1-2 and PLXND1 levels. GIPC3 expression was absent under all the experimental conditions assayed. Hence, for brevity, the corresponding Western blots are not shown. This figure is related to .

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Western Blot, Control, Infection, shRNA, Expressing

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Transgenic Assay, Plasmid Preparation, Mutagenesis, Derivative Assay, Selection, Stable Transfection, Knock-Out, Recombinant, Control, shRNA, Sequencing, Construct, Synthesized, Positive Control, Sterility, Concentration Assay

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-GIPC1 , Proteintech Group , Cat. #14822–1-AP. RRID: AB_2263269 , WB (1:3,000). This antibody detects GIPC1, GIPC2, and GIPC3 (our data).

Techniques: Sequencing