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: Human GPC4 targeting CRISPR/Cas9 (cat# sc-405200-NIC, Santa Cruz Biotechnology, Dallas, TX, USA), comprising a pair of human GPC4 targeted CRISPR/Cas9 knockout plasmids and its control plasmid (cat# sc-437281, Santa Cruz Biotechnology, Dallas, TX, USA) not targeting any specific gene (CRISPR control) were purchased from Santa Cruz Biotechnology.

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: Human GPC4 targeting CRISPR/Cas9 (cat# sc-405200-NIC, Santa Cruz Biotechnology, Dallas, TX, USA), comprising a pair of human GPC4 targeted CRISPR/Cas9 knockout plasmids and its control plasmid (cat# sc-437281, Santa Cruz Biotechnology, Dallas, TX, USA) not targeting any specific gene (CRISPR control) were purchased from Santa Cruz Biotechnology.

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: Human GPC4 targeting CRISPR/Cas9 (cat# sc-405200-NIC, Santa Cruz Biotechnology, Dallas, TX, USA), comprising a pair of human GPC4 targeted CRISPR/Cas9 knockout plasmids and its control plasmid (cat# sc-437281, Santa Cruz Biotechnology, Dallas, TX, USA) not targeting any specific gene (CRISPR control) were purchased from Santa Cruz Biotechnology.

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: Human GPC4 targeting CRISPR/Cas9 (cat# sc-405200-NIC, Santa Cruz Biotechnology, Dallas, TX, USA), comprising a pair of human GPC4 targeted CRISPR/Cas9 knockout plasmids and its control plasmid (cat# sc-437281, Santa Cruz Biotechnology, Dallas, TX, USA) not targeting any specific gene (CRISPR control) were purchased from Santa Cruz Biotechnology.

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: Human GPC4 targeting CRISPR/Cas9 (cat# sc-405200-NIC, Santa Cruz Biotechnology, Dallas, TX, USA), comprising a pair of human GPC4 targeted CRISPR/Cas9 knockout plasmids and its control plasmid (cat# sc-437281, Santa Cruz Biotechnology, Dallas, TX, USA) not targeting any specific gene (CRISPR control) were purchased from Santa Cruz Biotechnology.

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: Human GPC4 targeting CRISPR/Cas9 (cat# sc-405200-NIC, Santa Cruz Biotechnology, Dallas, TX, USA), comprising a pair of human GPC4 targeted CRISPR/Cas9 knockout plasmids and its control plasmid (cat# sc-437281, Santa Cruz Biotechnology, Dallas, TX, USA) not targeting any specific gene (CRISPR control) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing