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Santa Cruz Biotechnology cas9 control double nickase plasmid

( A ) RT-qPCR analysis to monitor efficacy of <t>CRISPR/Cas9-mediated</t> SLFN5 KO in PANC-1 (upper panel) and MIA-Pa-Ca-2 (lower panel) cells. Data are expressed as fold change over WT controls, and bar graphs represent mean ± SEM of 3 independent experiments. **** P < 0.0001. ( B ) Scatter plot (derived from high-throughput single-end RNA-seq analysis) showing the relationship between log 2 (fold change) (LFC) of 347 genes differentially expressed in both SLFN5 -KO ( x axis) and WT ( y axis) PANC-1 cells following IFN-α (5,000 IU for 6 hours) treatment. Select IFN-stimulated genes (ISGs) from the Reactome database ( https://www.reactome.org/content/detail/R-HSA-913531 ; identifier R-HAS-913531) are indicated. The red dashed line deviates from the unity line ( y = x , black line) and represents the linear regression fit ( y = 0.948 x + 0.026), capturing the overall trend between the 2 conditions. The slope ( m = 0.948) indicates a near one-to-one correspondence between the conditions, and the intercept ( c = 0.026) suggests minimal baseline difference. The high coefficient of determination ( R 2 = 0.89) reflects that 89% of the variance in WT LFCs is explained by SLFN5 -KO LFCs. ( C and D ) RT-qPCR analyses of relative mRNA expression of the indicated ISGs in SLFN5 -WT and -KO PANC-1 ( C ) and MIA-Pa-Ca-2 ( D ) cells untreated or treated with human IFN-α (5,000 IU, 6 hours). GAPDH was used for normalization and as an internal control. The data are expressed as fold change over the corresponding untreated cells; bar graphs represent mean ± SEM of 3 ( C ) or 4 ( D ) independent experiments. * P < 0.05. ( E ) PANC-1 SLFN5 -WT and-KO cells were stably transduced with an ISRE-luciferase promoter construct. Cells were incubated for 6 hours in the presence or absence of human IFN-α (5,000 IU) and luciferase activity was measured. Data are expressed as fold increase in luciferase activity in response to IFN-α treatment over control untreated samples for each condition. Bar graphs show mean ± SEM of 3 independent experiments. * P = 0.05. ( F ) ChIP for SLFN5 in PANC-1 cells transduced with lentivirus carrying doxycycline-inducible SLFN5-MYC-FLAG fusion construct. Cells were grown in the presence or absence of doxycycline for 48 hours, followed by IFN-α treatment for 6 hours. qPCR was performed on immunoprecipitated DNA with primers for the ISRE elements in the IFIT1 or ISG15 promoter. Primers for the RPL30 promoter were used as control. Data were normalized to their own IgG control and are expressed as fold enrichment over doxycycline-untreated cells. Shown are mean ± SEM of 3 independent experiments. * P < 0.05. Significance assessed by 2-tailed unpaired t test with Welch’s correction ( A ) or 1-tailed unpaired t test with Mann-Whitney test ( C – F ).

Cas9 Control Double Nickase Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma"

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

Journal: JCI Insight

doi: 10.1172/jci.insight.190031

( A ) RT-qPCR analysis to monitor efficacy of CRISPR/Cas9-mediated SLFN5 KO in PANC-1 (upper panel) and MIA-Pa-Ca-2 (lower panel) cells. Data are expressed as fold change over WT controls, and bar graphs represent mean ± SEM of 3 independent experiments. **** P < 0.0001. ( B ) Scatter plot (derived from high-throughput single-end RNA-seq analysis) showing the relationship between log 2 (fold change) (LFC) of 347 genes differentially expressed in both SLFN5 -KO ( x axis) and WT ( y axis) PANC-1 cells following IFN-α (5,000 IU for 6 hours) treatment. Select IFN-stimulated genes (ISGs) from the Reactome database ( https://www.reactome.org/content/detail/R-HSA-913531 ; identifier R-HAS-913531) are indicated. The red dashed line deviates from the unity line ( y = x , black line) and represents the linear regression fit ( y = 0.948 x + 0.026), capturing the overall trend between the 2 conditions. The slope ( m = 0.948) indicates a near one-to-one correspondence between the conditions, and the intercept ( c = 0.026) suggests minimal baseline difference. The high coefficient of determination ( R 2 = 0.89) reflects that 89% of the variance in WT LFCs is explained by SLFN5 -KO LFCs. ( C and D ) RT-qPCR analyses of relative mRNA expression of the indicated ISGs in SLFN5 -WT and -KO PANC-1 ( C ) and MIA-Pa-Ca-2 ( D ) cells untreated or treated with human IFN-α (5,000 IU, 6 hours). GAPDH was used for normalization and as an internal control. The data are expressed as fold change over the corresponding untreated cells; bar graphs represent mean ± SEM of 3 ( C ) or 4 ( D ) independent experiments. * P < 0.05. ( E ) PANC-1 SLFN5 -WT and-KO cells were stably transduced with an ISRE-luciferase promoter construct. Cells were incubated for 6 hours in the presence or absence of human IFN-α (5,000 IU) and luciferase activity was measured. Data are expressed as fold increase in luciferase activity in response to IFN-α treatment over control untreated samples for each condition. Bar graphs show mean ± SEM of 3 independent experiments. * P = 0.05. ( F ) ChIP for SLFN5 in PANC-1 cells transduced with lentivirus carrying doxycycline-inducible SLFN5-MYC-FLAG fusion construct. Cells were grown in the presence or absence of doxycycline for 48 hours, followed by IFN-α treatment for 6 hours. qPCR was performed on immunoprecipitated DNA with primers for the ISRE elements in the IFIT1 or ISG15 promoter. Primers for the RPL30 promoter were used as control. Data were normalized to their own IgG control and are expressed as fold enrichment over doxycycline-untreated cells. Shown are mean ± SEM of 3 independent experiments. * P < 0.05. Significance assessed by 2-tailed unpaired t test with Welch’s correction ( A ) or 1-tailed unpaired t test with Mann-Whitney test ( C – F ).

Figure Legend Snippet: ( A ) RT-qPCR analysis to monitor efficacy of CRISPR/Cas9-mediated SLFN5 KO in PANC-1 (upper panel) and MIA-Pa-Ca-2 (lower panel) cells. Data are expressed as fold change over WT controls, and bar graphs represent mean ± SEM of 3 independent experiments. **** P < 0.0001. ( B ) Scatter plot (derived from high-throughput single-end RNA-seq analysis) showing the relationship between log 2 (fold change) (LFC) of 347 genes differentially expressed in both SLFN5 -KO ( x axis) and WT ( y axis) PANC-1 cells following IFN-α (5,000 IU for 6 hours) treatment. Select IFN-stimulated genes (ISGs) from the Reactome database ( https://www.reactome.org/content/detail/R-HSA-913531 ; identifier R-HAS-913531) are indicated. The red dashed line deviates from the unity line ( y = x , black line) and represents the linear regression fit ( y = 0.948 x + 0.026), capturing the overall trend between the 2 conditions. The slope ( m = 0.948) indicates a near one-to-one correspondence between the conditions, and the intercept ( c = 0.026) suggests minimal baseline difference. The high coefficient of determination ( R 2 = 0.89) reflects that 89% of the variance in WT LFCs is explained by SLFN5 -KO LFCs. ( C and D ) RT-qPCR analyses of relative mRNA expression of the indicated ISGs in SLFN5 -WT and -KO PANC-1 ( C ) and MIA-Pa-Ca-2 ( D ) cells untreated or treated with human IFN-α (5,000 IU, 6 hours). GAPDH was used for normalization and as an internal control. The data are expressed as fold change over the corresponding untreated cells; bar graphs represent mean ± SEM of 3 ( C ) or 4 ( D ) independent experiments. * P < 0.05. ( E ) PANC-1 SLFN5 -WT and-KO cells were stably transduced with an ISRE-luciferase promoter construct. Cells were incubated for 6 hours in the presence or absence of human IFN-α (5,000 IU) and luciferase activity was measured. Data are expressed as fold increase in luciferase activity in response to IFN-α treatment over control untreated samples for each condition. Bar graphs show mean ± SEM of 3 independent experiments. * P = 0.05. ( F ) ChIP for SLFN5 in PANC-1 cells transduced with lentivirus carrying doxycycline-inducible SLFN5-MYC-FLAG fusion construct. Cells were grown in the presence or absence of doxycycline for 48 hours, followed by IFN-α treatment for 6 hours. qPCR was performed on immunoprecipitated DNA with primers for the ISRE elements in the IFIT1 or ISG15 promoter. Primers for the RPL30 promoter were used as control. Data were normalized to their own IgG control and are expressed as fold enrichment over doxycycline-untreated cells. Shown are mean ± SEM of 3 independent experiments. * P < 0.05. Significance assessed by 2-tailed unpaired t test with Welch’s correction ( A ) or 1-tailed unpaired t test with Mann-Whitney test ( C – F ).

Techniques Used: Quantitative RT-PCR, CRISPR, Derivative Assay, High Throughput Screening Assay, RNA Sequencing, Expressing, Control, Stable Transfection, Transduction, Luciferase, Construct, Incubation, Activity Assay, Immunoprecipitation, MANN-WHITNEY

( A – C ) RT-qPCR analysis to monitor ( A ) efficacy of CRISPR/Cas9-mediated Slfn5 disruption, ( B ) expression of Slfn5 in response to IFN-β treatment (5,000 IU for 6 hours) in Slfn5 WT cells, and ( C ) IFN-β–mediated (5,000 IU for 6 hours) induction of indicated murine ISGs in Slfn5 -WT and Slfn5 -KO cells. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over the corresponding controls, and the graphs represent mean ± SEM of 3 independent experiments. * P < 0.05, *** P < 0.001 by 2-tailed unpaired t test with Welch’s correction ( A and B ) or 1-tailed unpaired t test with Mann-Whitney test ( C ). ( D ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated in 6-well plates and counted on days 1, 2, and 3 after seeding. Data are mean of number of cells ± SEM of 3 independent experiments, each done in duplicate. Two-way repeated-measures ANOVA with Šidák’s multiple-comparison test; ** P < 0.01, **** P < 0.0001. ( E ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated into round-bottom 96-well plates under stem cell–permissive conditions to form 3D spheroids. After 7 days, spheres were imaged using a Cytation 3 cell imaging multi-mode reader to determine cross-sectional area. Data are expressed as percentages of WT parental spheres and represent mean ± SEM of 3 independent experiments, each done in triplicate. Two-tailed unpaired t test with Welch’s correction; * P ≤ 0.05.

Figure Legend Snippet: ( A – C ) RT-qPCR analysis to monitor ( A ) efficacy of CRISPR/Cas9-mediated Slfn5 disruption, ( B ) expression of Slfn5 in response to IFN-β treatment (5,000 IU for 6 hours) in Slfn5 WT cells, and ( C ) IFN-β–mediated (5,000 IU for 6 hours) induction of indicated murine ISGs in Slfn5 -WT and Slfn5 -KO cells. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over the corresponding controls, and the graphs represent mean ± SEM of 3 independent experiments. * P < 0.05, *** P < 0.001 by 2-tailed unpaired t test with Welch’s correction ( A and B ) or 1-tailed unpaired t test with Mann-Whitney test ( C ). ( D ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated in 6-well plates and counted on days 1, 2, and 3 after seeding. Data are mean of number of cells ± SEM of 3 independent experiments, each done in duplicate. Two-way repeated-measures ANOVA with Šidák’s multiple-comparison test; ** P < 0.01, **** P < 0.0001. ( E ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated into round-bottom 96-well plates under stem cell–permissive conditions to form 3D spheroids. After 7 days, spheres were imaged using a Cytation 3 cell imaging multi-mode reader to determine cross-sectional area. Data are expressed as percentages of WT parental spheres and represent mean ± SEM of 3 independent experiments, each done in triplicate. Two-tailed unpaired t test with Welch’s correction; * P ≤ 0.05.

Techniques Used: Quantitative RT-PCR, CRISPR, Disruption, Expressing, Control, MANN-WHITNEY, Comparison, Imaging, Two Tailed Test

Control:

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma
Article Snippet: .. Briefly, 2 μg of the Cas9 Control Double Nickase plasmid (Santa Cruz Biotechnology, sc437281) or 2 μg of the Slfn5 Double Nickase plasmid (Santa Cruz Biotechnology, sc435875-NIC) were transfected into murine luciferase-expressing KPC1199 cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. ..

Plasmid Preparation:

Transfection:

Luciferase:

Image Search Results

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A ) RT-qPCR analysis to monitor efficacy of CRISPR/Cas9-mediated SLFN5 KO in PANC-1 (upper panel) and MIA-Pa-Ca-2 (lower panel) cells. Data are expressed as fold change over WT controls, and bar graphs represent mean ± SEM of 3 independent experiments. **** P < 0.0001. ( B ) Scatter plot (derived from high-throughput single-end RNA-seq analysis) showing the relationship between log 2 (fold change) (LFC) of 347 genes differentially expressed in both SLFN5 -KO ( x axis) and WT ( y axis) PANC-1 cells following IFN-α (5,000 IU for 6 hours) treatment. Select IFN-stimulated genes (ISGs) from the Reactome database ( https://www.reactome.org/content/detail/R-HSA-913531 ; identifier R-HAS-913531) are indicated. The red dashed line deviates from the unity line ( y = x , black line) and represents the linear regression fit ( y = 0.948 x + 0.026), capturing the overall trend between the 2 conditions. The slope ( m = 0.948) indicates a near one-to-one correspondence between the conditions, and the intercept ( c = 0.026) suggests minimal baseline difference. The high coefficient of determination ( R 2 = 0.89) reflects that 89% of the variance in WT LFCs is explained by SLFN5 -KO LFCs. ( C and D ) RT-qPCR analyses of relative mRNA expression of the indicated ISGs in SLFN5 -WT and -KO PANC-1 ( C ) and MIA-Pa-Ca-2 ( D ) cells untreated or treated with human IFN-α (5,000 IU, 6 hours). GAPDH was used for normalization and as an internal control. The data are expressed as fold change over the corresponding untreated cells; bar graphs represent mean ± SEM of 3 ( C ) or 4 ( D ) independent experiments. * P < 0.05. ( E ) PANC-1 SLFN5 -WT and-KO cells were stably transduced with an ISRE-luciferase promoter construct. Cells were incubated for 6 hours in the presence or absence of human IFN-α (5,000 IU) and luciferase activity was measured. Data are expressed as fold increase in luciferase activity in response to IFN-α treatment over control untreated samples for each condition. Bar graphs show mean ± SEM of 3 independent experiments. * P = 0.05. ( F ) ChIP for SLFN5 in PANC-1 cells transduced with lentivirus carrying doxycycline-inducible SLFN5-MYC-FLAG fusion construct. Cells were grown in the presence or absence of doxycycline for 48 hours, followed by IFN-α treatment for 6 hours. qPCR was performed on immunoprecipitated DNA with primers for the ISRE elements in the IFIT1 or ISG15 promoter. Primers for the RPL30 promoter were used as control. Data were normalized to their own IgG control and are expressed as fold enrichment over doxycycline-untreated cells. Shown are mean ± SEM of 3 independent experiments. * P < 0.05. Significance assessed by 2-tailed unpaired t test with Welch’s correction ( A ) or 1-tailed unpaired t test with Mann-Whitney test ( C – F ).

Article Snippet: Briefly, 2 μg of the Cas9 Control Double Nickase plasmid (Santa Cruz Biotechnology, sc437281) or 2 μg of the Slfn5 Double Nickase plasmid (Santa Cruz Biotechnology, sc435875-NIC) were transfected into murine luciferase-expressing KPC1199 cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, CRISPR, Derivative Assay, High Throughput Screening Assay, RNA Sequencing, Expressing, Control, Stable Transfection, Transduction, Luciferase, Construct, Incubation, Activity Assay, Immunoprecipitation, MANN-WHITNEY

Journal: JCI Insight

Article Title: Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma

doi: 10.1172/jci.insight.190031

Figure Lengend Snippet: ( A – C ) RT-qPCR analysis to monitor ( A ) efficacy of CRISPR/Cas9-mediated Slfn5 disruption, ( B ) expression of Slfn5 in response to IFN-β treatment (5,000 IU for 6 hours) in Slfn5 WT cells, and ( C ) IFN-β–mediated (5,000 IU for 6 hours) induction of indicated murine ISGs in Slfn5 -WT and Slfn5 -KO cells. The expression levels of the indicated genes were determined using Gapdh for normalization and as an internal control. The data are expressed as fold change over the corresponding controls, and the graphs represent mean ± SEM of 3 independent experiments. * P < 0.05, *** P < 0.001 by 2-tailed unpaired t test with Welch’s correction ( A and B ) or 1-tailed unpaired t test with Mann-Whitney test ( C ). ( D ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated in 6-well plates and counted on days 1, 2, and 3 after seeding. Data are mean of number of cells ± SEM of 3 independent experiments, each done in duplicate. Two-way repeated-measures ANOVA with Šidák’s multiple-comparison test; ** P < 0.01, **** P < 0.0001. ( E ) Slfn5 -WT and Slfn5 -KO KPC1199 cells were plated into round-bottom 96-well plates under stem cell–permissive conditions to form 3D spheroids. After 7 days, spheres were imaged using a Cytation 3 cell imaging multi-mode reader to determine cross-sectional area. Data are expressed as percentages of WT parental spheres and represent mean ± SEM of 3 independent experiments, each done in triplicate. Two-tailed unpaired t test with Welch’s correction; * P ≤ 0.05.

Techniques: Quantitative RT-PCR, CRISPR, Disruption, Expressing, Control, MANN-WHITNEY, Comparison, Imaging, Two Tailed Test

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.

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

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 .

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 .

Techniques: Expressing

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.

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.

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

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.

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.

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

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).

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).

Techniques: Quantitative Proteomics, Expressing, Migration, Gene Expression, Activation Assay, Inhibition, Activity Assay

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).

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).

Techniques: Gene Expression

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cas9 control double nickase plasmid (Santa Cruz Biotechnology)

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1) Product Images from "Schlafen 5 is an intracellular immune checkpoint and controls IFN responses in pancreatic ductal adenocarcinoma"

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