Structured Review

OriGene cdna
Increased expression of <t>CRRY</t> in the RPE cells by subretinal injection of AAV-CRRY. ( A ) Histogram showing relative CRRY, DAF1, DAF2, CD59a, CD59b, and CFH mRNA levels by qRT-PCR. Each mRNA level was normalized to 18S rRNA. ( B ) Representative immunoblot of CRRY (65-kDa isoform), Myc, and β-actin using RPE homogenate (10 μg of protein per lane). Protein and <t>cDNA</t> samples were obtained from 1-y-old Abca4 −/− mice injected with AAV-CRRY and -null viruses. Data represent mean ± SD; n = 5 mice. Each protein and cDNA sample was run in triplicate. M.W., molecular weight.
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Images

1) Product Images from "Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease"

Article Title: Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1620299114

Increased expression of CRRY in the RPE cells by subretinal injection of AAV-CRRY. ( A ) Histogram showing relative CRRY, DAF1, DAF2, CD59a, CD59b, and CFH mRNA levels by qRT-PCR. Each mRNA level was normalized to 18S rRNA. ( B ) Representative immunoblot of CRRY (65-kDa isoform), Myc, and β-actin using RPE homogenate (10 μg of protein per lane). Protein and cDNA samples were obtained from 1-y-old Abca4 −/− mice injected with AAV-CRRY and -null viruses. Data represent mean ± SD; n = 5 mice. Each protein and cDNA sample was run in triplicate. M.W., molecular weight.
Figure Legend Snippet: Increased expression of CRRY in the RPE cells by subretinal injection of AAV-CRRY. ( A ) Histogram showing relative CRRY, DAF1, DAF2, CD59a, CD59b, and CFH mRNA levels by qRT-PCR. Each mRNA level was normalized to 18S rRNA. ( B ) Representative immunoblot of CRRY (65-kDa isoform), Myc, and β-actin using RPE homogenate (10 μg of protein per lane). Protein and cDNA samples were obtained from 1-y-old Abca4 −/− mice injected with AAV-CRRY and -null viruses. Data represent mean ± SD; n = 5 mice. Each protein and cDNA sample was run in triplicate. M.W., molecular weight.

Techniques Used: Expressing, Injection, Quantitative RT-PCR, Mouse Assay, Molecular Weight

AAV-CRRY treatment does not change the oxidative stress level in Abca4 −/− mice. ( A ) Histogram showing the relative SOD-1 and SOD-2 mRNA levels by qRT-PCR. Each mRNA level was normalized to 18S rRNA. ( B ) Representative immunoblots of SOD-1, SOD-2, and α-tubulin using RPE homogenate (10 μg of protein per lane). ( C ) Histogram showing SOD-1 and SOD-2 normalized protein data in the RPE homogenate samples shown in B . Protein and cDNA samples were obtained from 7-mo-old Abca4 −/− mice injected with AAV-CRRY and -null viruses ( n = 4). Each protein and cDNA sample was run in triplicate.
Figure Legend Snippet: AAV-CRRY treatment does not change the oxidative stress level in Abca4 −/− mice. ( A ) Histogram showing the relative SOD-1 and SOD-2 mRNA levels by qRT-PCR. Each mRNA level was normalized to 18S rRNA. ( B ) Representative immunoblots of SOD-1, SOD-2, and α-tubulin using RPE homogenate (10 μg of protein per lane). ( C ) Histogram showing SOD-1 and SOD-2 normalized protein data in the RPE homogenate samples shown in B . Protein and cDNA samples were obtained from 7-mo-old Abca4 −/− mice injected with AAV-CRRY and -null viruses ( n = 4). Each protein and cDNA sample was run in triplicate.

Techniques Used: Mouse Assay, Quantitative RT-PCR, Western Blot, Injection

2) Product Images from "The protein kinase Pak4 disrupts mammary acinar architecture and promotes mammary tumorigenesis"

Article Title: The protein kinase Pak4 disrupts mammary acinar architecture and promotes mammary tumorigenesis

Journal: Oncogene

doi: 10.1038/onc.2010.329

Pak4 is overexpressed in many different types of human tumors and cancer cell lines. A . Quantitative PCR analysis of Pak4 gene expression using cDNA from different types of human tumor tissues and normal tissue. Data are represented as the ratio normalized to β-actin gene expression. 1, Breast. 2, Endometrium. 3, Esophagus. 4, Ovary. 5, Prostate. 6, Urinary bladder. 7, Testis. B . Western blot analysis illustrates high levels of Pak4 expression in human cancer cell lines compared to normal cell lines. The following cell lines were analyzed: Colon cancer cell lines (HT29 and HCT116), prostate cancer cell lines (LNCap, CWR, DU145, DUPro, and PC-3), and normal small intestine and prostate cell lines (INT407 and RWPE-1). C . Pak4 is overexpressed in response to oncogene expression in iMMECs. In B and C, 40 μg of protein extract was used; β-actin served as a loading control.
Figure Legend Snippet: Pak4 is overexpressed in many different types of human tumors and cancer cell lines. A . Quantitative PCR analysis of Pak4 gene expression using cDNA from different types of human tumor tissues and normal tissue. Data are represented as the ratio normalized to β-actin gene expression. 1, Breast. 2, Endometrium. 3, Esophagus. 4, Ovary. 5, Prostate. 6, Urinary bladder. 7, Testis. B . Western blot analysis illustrates high levels of Pak4 expression in human cancer cell lines compared to normal cell lines. The following cell lines were analyzed: Colon cancer cell lines (HT29 and HCT116), prostate cancer cell lines (LNCap, CWR, DU145, DUPro, and PC-3), and normal small intestine and prostate cell lines (INT407 and RWPE-1). C . Pak4 is overexpressed in response to oncogene expression in iMMECs. In B and C, 40 μg of protein extract was used; β-actin served as a loading control.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot

3) Product Images from "p53's choice of myocardial death or survival: Oxygen protects infarct myocardium by recruiting p53 on NOS3 promoter through regulation of p53-Lys118 acetylation"

Article Title: p53's choice of myocardial death or survival: Oxygen protects infarct myocardium by recruiting p53 on NOS3 promoter through regulation of p53-Lys118 acetylation

Journal: EMBO Molecular Medicine

doi: 10.1002/emmm.201202055

Oxygenation inhibits acetylation of p53-Lys 118 residue and increases survival of rat neonatal cardiomyocyte (RNC) cells Western-blot results show the abolition of p53 expression in RNC cells (lane 2), while addition of p53 Lys118-Ala118 cDNA and p53 Wt cDNA to RNC p53 −/− cells show the expression of p53 (lanes 3 and 4). Control cells show expression of p53 (lane 1). Immunoprecipitation results show acetylation of p53 at Lys118 residue in RNC cells under SD conditions (lane 2). However, this expression is abolished upon oxygenation (lane 3). Acetylation at p53 Lys118 is absent in control, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells under both SD and SD + Oxy conditions (lanes 4–9). Cell survival analysis of control, SD and SD + Oxy-treated RNC, RNC + p53 siRNA, RNC + p53 siRNA + p53-Lys 118 (Mut) cDNA and RNC + p53 siRNA + Wt p53 cDNA cells show an increase in the SD-treated RNC + p53 siRNA and RNC + p53 siRNA + p53-Lys 118 (Mut) cDNA cells compared to RNC cells, exhibiting the crucial role of deacetylation of p53 Lys 118 in cell survival. Data represent mean ± SD of eight independent measurements. * p = 2.6E−14 versus respective SD group; # p = 9.3E−08 versus respective SD group. Luciferase assay shows activation of BAX-p53-RE and NOS3-p53-RE in SD and SD + Oxy-treated RNC cells. Neither BAX nor NOS3 is activated in RNC + p53 siRNA cells. Oxygenation caused a switch of BAX activation to NOS3 activation in RNC p53-Lys 118 (Mut) cells. Data represent mean ± SD of eight independent measurements. ChIP assay shows the binding of p53 to its respective NOS3 and BAX RE in RNC cells (lanes 4 and 5). However, no binding of p53 to BAX-RE or NOS3-RE is observed in RNC p53 −/− cells (lanes 7 and 8). However, p53 binds to NOS3-RE in SD + Oxy-treated RNC p53-Lys 118 (Mut) cells (lane 11). These results suggest the importance of p53 Lys118 acetylation in activation of either BAX-RE or NOS3-RE and regulation of apoptotic or survival pathway respectively. Similar results were observed at mRNA and protein level using RT-PCR and Western-blot techniques in RNC, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells. Expression of p53 downstream genes in RNC, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells. SD-treated RNC cells show activation of apoptotic genes whereas RNC p53 −/− and RNC p53-Lys 118 (Mut) cells show no involvement in apoptotic gene activation.
Figure Legend Snippet: Oxygenation inhibits acetylation of p53-Lys 118 residue and increases survival of rat neonatal cardiomyocyte (RNC) cells Western-blot results show the abolition of p53 expression in RNC cells (lane 2), while addition of p53 Lys118-Ala118 cDNA and p53 Wt cDNA to RNC p53 −/− cells show the expression of p53 (lanes 3 and 4). Control cells show expression of p53 (lane 1). Immunoprecipitation results show acetylation of p53 at Lys118 residue in RNC cells under SD conditions (lane 2). However, this expression is abolished upon oxygenation (lane 3). Acetylation at p53 Lys118 is absent in control, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells under both SD and SD + Oxy conditions (lanes 4–9). Cell survival analysis of control, SD and SD + Oxy-treated RNC, RNC + p53 siRNA, RNC + p53 siRNA + p53-Lys 118 (Mut) cDNA and RNC + p53 siRNA + Wt p53 cDNA cells show an increase in the SD-treated RNC + p53 siRNA and RNC + p53 siRNA + p53-Lys 118 (Mut) cDNA cells compared to RNC cells, exhibiting the crucial role of deacetylation of p53 Lys 118 in cell survival. Data represent mean ± SD of eight independent measurements. * p = 2.6E−14 versus respective SD group; # p = 9.3E−08 versus respective SD group. Luciferase assay shows activation of BAX-p53-RE and NOS3-p53-RE in SD and SD + Oxy-treated RNC cells. Neither BAX nor NOS3 is activated in RNC + p53 siRNA cells. Oxygenation caused a switch of BAX activation to NOS3 activation in RNC p53-Lys 118 (Mut) cells. Data represent mean ± SD of eight independent measurements. ChIP assay shows the binding of p53 to its respective NOS3 and BAX RE in RNC cells (lanes 4 and 5). However, no binding of p53 to BAX-RE or NOS3-RE is observed in RNC p53 −/− cells (lanes 7 and 8). However, p53 binds to NOS3-RE in SD + Oxy-treated RNC p53-Lys 118 (Mut) cells (lane 11). These results suggest the importance of p53 Lys118 acetylation in activation of either BAX-RE or NOS3-RE and regulation of apoptotic or survival pathway respectively. Similar results were observed at mRNA and protein level using RT-PCR and Western-blot techniques in RNC, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells. Expression of p53 downstream genes in RNC, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells. SD-treated RNC cells show activation of apoptotic genes whereas RNC p53 −/− and RNC p53-Lys 118 (Mut) cells show no involvement in apoptotic gene activation.

Techniques Used: Western Blot, Expressing, Immunoprecipitation, Luciferase, Activation Assay, Chromatin Immunoprecipitation, Binding Assay, Reverse Transcription Polymerase Chain Reaction

TIP60 regulates p53-mediated activation of BAX or NOS3 gene in H9c2, H9c2 p53 −/− and H9c2 p53-Lys 118 (Mut) cells TIP60 protein expression in H9c2 cells under control, SD, and SD + Oxy conditions. SD induces TIP60 expression, which is inhibited by oxygenation. TIP60 expression in MI hearts is inhibited by oxygenation. Co-immunoprecipitation of p53 with TIP60 shows that TIP60 binds to p53 only in MI hearts and this interaction is abolished upon oxygenation. Input and SD-treated H9c2 cells with TIP60 gene-silencing were used as controls. Effect of TIP60 on the survival of SD H9c2 cells. Silencing of TIP60 using TIP60 siRNA in H9c2, H9c2 p53 −/− and H9c2 p53-Lys 118 (Mut) cells increases the survival of SD cells. Addition of TIP60 using cDNA results decreases the survival of SD cells. Data represent mean ± SD of four independent measurements. Effect of TIP60 on the binding of p53 to BAX-RE and NOS3-RE was analysed using ChIP in H9c2 cells. Results show that TIP60 gene-silencing results in binding of p53 to NOS3-RE in both SD and SD + Oxy cells. Similarly, exogenous addition of TIP60 cDNA results in binding of p53 to BAX-RE in both SD and SD + Oxy cells. Effect of TIP60 siRNA and TIP60 cDNA addition on p53-Lys 118 acetylation, NOS3 and BAX protein expression was determined using Western blot. Data show that TIP60 siRNA abolishes p53-Lys 118 acetylation, increases the expression of NOS3 protein and decreases BAX expression in H9c2 cells. Similarly, TIP60 cDNA increases p53-Lys 118 acetylation, decreases the expression of NOS3 protein and increased BAX expression in H9c2 cells. Expression of p53 downstream genes was determined in the SD and SD + Oxy group of H9c2 cells in presence and absence of TIP60 siRNA and TIP60 cDNA. Data show that TIP60 cDNA increases the expression of these genes and TIP60 siRNA decreases their expression in both SD and SD + Oxy groups.
Figure Legend Snippet: TIP60 regulates p53-mediated activation of BAX or NOS3 gene in H9c2, H9c2 p53 −/− and H9c2 p53-Lys 118 (Mut) cells TIP60 protein expression in H9c2 cells under control, SD, and SD + Oxy conditions. SD induces TIP60 expression, which is inhibited by oxygenation. TIP60 expression in MI hearts is inhibited by oxygenation. Co-immunoprecipitation of p53 with TIP60 shows that TIP60 binds to p53 only in MI hearts and this interaction is abolished upon oxygenation. Input and SD-treated H9c2 cells with TIP60 gene-silencing were used as controls. Effect of TIP60 on the survival of SD H9c2 cells. Silencing of TIP60 using TIP60 siRNA in H9c2, H9c2 p53 −/− and H9c2 p53-Lys 118 (Mut) cells increases the survival of SD cells. Addition of TIP60 using cDNA results decreases the survival of SD cells. Data represent mean ± SD of four independent measurements. Effect of TIP60 on the binding of p53 to BAX-RE and NOS3-RE was analysed using ChIP in H9c2 cells. Results show that TIP60 gene-silencing results in binding of p53 to NOS3-RE in both SD and SD + Oxy cells. Similarly, exogenous addition of TIP60 cDNA results in binding of p53 to BAX-RE in both SD and SD + Oxy cells. Effect of TIP60 siRNA and TIP60 cDNA addition on p53-Lys 118 acetylation, NOS3 and BAX protein expression was determined using Western blot. Data show that TIP60 siRNA abolishes p53-Lys 118 acetylation, increases the expression of NOS3 protein and decreases BAX expression in H9c2 cells. Similarly, TIP60 cDNA increases p53-Lys 118 acetylation, decreases the expression of NOS3 protein and increased BAX expression in H9c2 cells. Expression of p53 downstream genes was determined in the SD and SD + Oxy group of H9c2 cells in presence and absence of TIP60 siRNA and TIP60 cDNA. Data show that TIP60 cDNA increases the expression of these genes and TIP60 siRNA decreases their expression in both SD and SD + Oxy groups.

Techniques Used: Activation Assay, Expressing, Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation, Western Blot

TIP60 regulates p53-mediated activation of BAX or NOS3 gene in RNC, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells Western-blot analysis was conducted to study the expression of TIP60 in control, SD and SD + Oxy-treated cells. SD induces TIP60 expression (lane 2); however, this expression is significantly inhibited by oxygenation (lane 3). Western blots of TIP60 expression in MI and MI + Oxy-treated cardiac tissue. Results show increased TIP60 expression in MI tissue (lane 2); however, this expression is significantly reduced in MI + Oxy-treated cardiac tissue (lane 3). Co-immunoprecipitation study was conducted to study p53 and TIP60 interaction in MI and MI + Oxy-treated RNC cells. Results show p53 interaction with TIP60 in SD-treated cells (lane 4); however, this oxygenation significantly inhibits this interaction (lane 5) in RNC cells. Input and TIP60 siRNA were used as controls for the study. The role of TIP60 in cell s urvival was studied in RNC cells using flow cytometry. Transfection with TIP60 siRNA results in increase in cell-survival fraction in RNC cells while TIP60 cDNA results in a decrease in cell-survival fraction. Conversely, neither TIP60 siRNA nor TIP60 cDNA has any effect on cell survival in RNC p53 −/− and RNC p53-Lys 118 (Mut) cells. Data represent mean ± SD of four independent measurements. CHIP assay was conducted to study the role of TIP60 in p53 binding to BAX-RE or NOS3-RE in SD and SD + Oxy-treated RNC cells. Results show that transfection with TIP60 siRNA results in p53 binding to NOS-RE in both SD and SD + Oxy-treated cells. However, exogenous addition of TIP60 cDNA result in p53 binding to BAX-RE in both SDF and SD + Oxy-treated cells. Effect of TIP60 siRNA and TIP60 cDNA addition on p53-Lys 118 acetylation, NOS3 and BAX protein expression was studied in SD and SD + Oxy-treated RNC cells. Transfection with TIP60 siRNA led to abolition of p53-Lys 118 acetylation, increased NOS3 expression and decreased BAX expression in RNC cells. Contrarily, transfection with TIP60 cDNA led to increased p53-Lys 118 acetylation, decreased NOS3 expression and increased BAX expression in RNC cells. Gene array was studied to study the role of TIP60 in activation of p53 downstream genes in SD and SD + Oxy treated cells. Transfection with TIP60 cDNA results in the activations of apoptotic genes in SD and SD + Oxy-treated cells when compared to TIP60 siRNA-transfected SD and SD + Oxy-treated groups.
Figure Legend Snippet: TIP60 regulates p53-mediated activation of BAX or NOS3 gene in RNC, RNC p53 −/− and RNC p53-Lys 118 (Mut) cells Western-blot analysis was conducted to study the expression of TIP60 in control, SD and SD + Oxy-treated cells. SD induces TIP60 expression (lane 2); however, this expression is significantly inhibited by oxygenation (lane 3). Western blots of TIP60 expression in MI and MI + Oxy-treated cardiac tissue. Results show increased TIP60 expression in MI tissue (lane 2); however, this expression is significantly reduced in MI + Oxy-treated cardiac tissue (lane 3). Co-immunoprecipitation study was conducted to study p53 and TIP60 interaction in MI and MI + Oxy-treated RNC cells. Results show p53 interaction with TIP60 in SD-treated cells (lane 4); however, this oxygenation significantly inhibits this interaction (lane 5) in RNC cells. Input and TIP60 siRNA were used as controls for the study. The role of TIP60 in cell s urvival was studied in RNC cells using flow cytometry. Transfection with TIP60 siRNA results in increase in cell-survival fraction in RNC cells while TIP60 cDNA results in a decrease in cell-survival fraction. Conversely, neither TIP60 siRNA nor TIP60 cDNA has any effect on cell survival in RNC p53 −/− and RNC p53-Lys 118 (Mut) cells. Data represent mean ± SD of four independent measurements. CHIP assay was conducted to study the role of TIP60 in p53 binding to BAX-RE or NOS3-RE in SD and SD + Oxy-treated RNC cells. Results show that transfection with TIP60 siRNA results in p53 binding to NOS-RE in both SD and SD + Oxy-treated cells. However, exogenous addition of TIP60 cDNA result in p53 binding to BAX-RE in both SDF and SD + Oxy-treated cells. Effect of TIP60 siRNA and TIP60 cDNA addition on p53-Lys 118 acetylation, NOS3 and BAX protein expression was studied in SD and SD + Oxy-treated RNC cells. Transfection with TIP60 siRNA led to abolition of p53-Lys 118 acetylation, increased NOS3 expression and decreased BAX expression in RNC cells. Contrarily, transfection with TIP60 cDNA led to increased p53-Lys 118 acetylation, decreased NOS3 expression and increased BAX expression in RNC cells. Gene array was studied to study the role of TIP60 in activation of p53 downstream genes in SD and SD + Oxy treated cells. Transfection with TIP60 cDNA results in the activations of apoptotic genes in SD and SD + Oxy-treated cells when compared to TIP60 siRNA-transfected SD and SD + Oxy-treated groups.

Techniques Used: Activation Assay, Western Blot, Expressing, Immunoprecipitation, Flow Cytometry, Cytometry, Transfection, Chromatin Immunoprecipitation, Binding Assay

4) Product Images from "MUC13 contributes to rewiring of glucose metabolism in pancreatic cancer"

Article Title: MUC13 contributes to rewiring of glucose metabolism in pancreatic cancer

Journal: Oncogenesis

doi: 10.1038/s41389-018-0031-0

MUC13 and Glut-1 expression directly correlate in human pancreatic cancer tissues. a qPCR was performed to analyze the levels of MUC13 and Glut-1 using RNA from freshly collected human PDAC tissues ( N = 8) procured from Baptist Memorial hospital, Memphis. b and cDNA from PDAC tissues ( N = 6) procured from Origene. The correlation coefficient ( r ) and coefficient of determinations ( r 2 ) were 0.982 and 0.862, respectively. The analysis was performed by graph pad prism software. c Schematic representation showing the overall mechanism of MUC13 in modulating glucose metabolic network and driving tumorigenic microenvironment
Figure Legend Snippet: MUC13 and Glut-1 expression directly correlate in human pancreatic cancer tissues. a qPCR was performed to analyze the levels of MUC13 and Glut-1 using RNA from freshly collected human PDAC tissues ( N = 8) procured from Baptist Memorial hospital, Memphis. b and cDNA from PDAC tissues ( N = 6) procured from Origene. The correlation coefficient ( r ) and coefficient of determinations ( r 2 ) were 0.982 and 0.862, respectively. The analysis was performed by graph pad prism software. c Schematic representation showing the overall mechanism of MUC13 in modulating glucose metabolic network and driving tumorigenic microenvironment

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Software

5) Product Images from "Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils *"

Article Title: Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils *

Journal:

doi: 10.1074/jbc.M806194200

Expression of MBOAT mRNAs in human neutrophils and production of human MBOAT proteins in ale1 Δ yeast. A , expression of human MBOAT mRNAs was examined using cDNA prepared from total human neutrophil RNA by PCR with primer pairs specific for
Figure Legend Snippet: Expression of MBOAT mRNAs in human neutrophils and production of human MBOAT proteins in ale1 Δ yeast. A , expression of human MBOAT mRNAs was examined using cDNA prepared from total human neutrophil RNA by PCR with primer pairs specific for

Techniques Used: Expressing, Polymerase Chain Reaction

6) Product Images from "MUC13 contributes to rewiring of glucose metabolism in pancreatic cancer"

Article Title: MUC13 contributes to rewiring of glucose metabolism in pancreatic cancer

Journal: Oncogenesis

doi: 10.1038/s41389-018-0031-0

MUC13 and Glut-1 expression directly correlate in human pancreatic cancer tissues. a qPCR was performed to analyze the levels of MUC13 and Glut-1 using RNA from freshly collected human PDAC tissues ( N = 8) procured from Baptist Memorial hospital, Memphis. b and cDNA from PDAC tissues ( N = 6) procured from Origene. The correlation coefficient ( r ) and coefficient of determinations ( r 2 ) were 0.982 and 0.862, respectively. The analysis was performed by graph pad prism software. c Schematic representation showing the overall mechanism of MUC13 in modulating glucose metabolic network and driving tumorigenic microenvironment
Figure Legend Snippet: MUC13 and Glut-1 expression directly correlate in human pancreatic cancer tissues. a qPCR was performed to analyze the levels of MUC13 and Glut-1 using RNA from freshly collected human PDAC tissues ( N = 8) procured from Baptist Memorial hospital, Memphis. b and cDNA from PDAC tissues ( N = 6) procured from Origene. The correlation coefficient ( r ) and coefficient of determinations ( r 2 ) were 0.982 and 0.862, respectively. The analysis was performed by graph pad prism software. c Schematic representation showing the overall mechanism of MUC13 in modulating glucose metabolic network and driving tumorigenic microenvironment

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Software

7) Product Images from "Cellular Functions of the Autism Risk Factor PTCHD1 in Mice"

Article Title: Cellular Functions of the Autism Risk Factor PTCHD1 in Mice

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1393-17.2017

Ptchd1 KO mouse generation and validation. A , Ptchd1 KO strategy, in which the Ptchd1 gene was modified by replacing exon 2 with a cassette containing a gene trap lacZ-Neo cassette (engrailed 2 splicing acceptor) flanked by Frt sites and additional loxP sites flanking Exon 2. Conditional Ptchd1 floxed animals were obtained by breeding to Flip deleter mice, resulting in an excision of the trap cassette. The Ptchd1 KO allele was generated by crossing Ptchd1 floxed animals with CMVcre deleter mice. B , RT-PCR of WT and Ptchd1 −/y cDNA showing successful removal of exon 2 and splicing of exon 1 into exon 3, resulting into a frame shift. Splice junctions were confirmed by DNA sequencing. C , Specificity of a Ptchd1-specific antibody was confirmed by Western blot analysis of HEK293T cells overexpressing a Ptchd1-EGFP fusion protein. D , Successful ablation of Ptchd1 protein in KO animals was verified by Western blot analysis of hippocampal tissue lysate (left) and cerebellar tissue lysate (middle). Right, Ptchd1 protein relative expression levels across brain regions in adult P60 mouse. Similar results were obtained in three independent experiments.
Figure Legend Snippet: Ptchd1 KO mouse generation and validation. A , Ptchd1 KO strategy, in which the Ptchd1 gene was modified by replacing exon 2 with a cassette containing a gene trap lacZ-Neo cassette (engrailed 2 splicing acceptor) flanked by Frt sites and additional loxP sites flanking Exon 2. Conditional Ptchd1 floxed animals were obtained by breeding to Flip deleter mice, resulting in an excision of the trap cassette. The Ptchd1 KO allele was generated by crossing Ptchd1 floxed animals with CMVcre deleter mice. B , RT-PCR of WT and Ptchd1 −/y cDNA showing successful removal of exon 2 and splicing of exon 1 into exon 3, resulting into a frame shift. Splice junctions were confirmed by DNA sequencing. C , Specificity of a Ptchd1-specific antibody was confirmed by Western blot analysis of HEK293T cells overexpressing a Ptchd1-EGFP fusion protein. D , Successful ablation of Ptchd1 protein in KO animals was verified by Western blot analysis of hippocampal tissue lysate (left) and cerebellar tissue lysate (middle). Right, Ptchd1 protein relative expression levels across brain regions in adult P60 mouse. Similar results were obtained in three independent experiments.

Techniques Used: Modification, Mouse Assay, Generated, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Western Blot, Expressing

8) Product Images from "Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils *"

Article Title: Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils *

Journal:

doi: 10.1074/jbc.M806194200

Expression of MBOAT mRNAs in human neutrophils and production of human MBOAT proteins in ale1 Δ yeast. A , expression of human MBOAT mRNAs was examined using cDNA prepared from total human neutrophil RNA by PCR with primer pairs specific for
Figure Legend Snippet: Expression of MBOAT mRNAs in human neutrophils and production of human MBOAT proteins in ale1 Δ yeast. A , expression of human MBOAT mRNAs was examined using cDNA prepared from total human neutrophil RNA by PCR with primer pairs specific for

Techniques Used: Expressing, Polymerase Chain Reaction

9) Product Images from "Overexpression of MicroRNAs from the miR-17-92 Paralog Clusters in AIDS-Related Non-Hodgkin's Lymphomas"

Article Title: Overexpression of MicroRNAs from the miR-17-92 Paralog Clusters in AIDS-Related Non-Hodgkin's Lymphomas

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020781

p21 protein and RNA expression in primary AIDS-NHL samples. ( A ) p21 Western blot analysis of primary AIDS- BL and AIDS-DLBCL samples. The blot was stripped and sequentially probed for p53 (Ab clone DO-7) and β-actin. No p21 protein expression was detected. The positive control is lysate from p21 cDNA transfected B cell line Ramos. ( B ) Taqman q-PCR of p21 mRNA levels in primary AIDS-NHL samples shown in A. In each sample, p21 mRNA level is shown as a % of β-actin expression, which is set to 100.
Figure Legend Snippet: p21 protein and RNA expression in primary AIDS-NHL samples. ( A ) p21 Western blot analysis of primary AIDS- BL and AIDS-DLBCL samples. The blot was stripped and sequentially probed for p53 (Ab clone DO-7) and β-actin. No p21 protein expression was detected. The positive control is lysate from p21 cDNA transfected B cell line Ramos. ( B ) Taqman q-PCR of p21 mRNA levels in primary AIDS-NHL samples shown in A. In each sample, p21 mRNA level is shown as a % of β-actin expression, which is set to 100.

Techniques Used: RNA Expression, Western Blot, Expressing, Positive Control, Transfection, Polymerase Chain Reaction

Related Articles

Clone Assay:

Article Title: Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils *
Article Snippet: .. Cloning and Expression of Human MBOAT Proteins in the Yeast ale1 Δ Mutant —Plasmids containing cDNA coding for human MBOAT1 (GenBank™ accession number NM_ 001080480.1), MBOAT2 (GenBank accession number ), and MBOAT5 (GenBank accession number ) were purchased from Origene (Rockville, MD), and a cDNA clone for MBOAT7 (GenBank accession number ) was purchased from Open Biosystems (Huntsville, AL). .. The open reading frames were subcloned into the pYES2.1-TOPO vector according to the manufacturer's instructions.

Amplification:

Article Title: Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease
Article Snippet: .. The murine CRRY gene was amplified by PCR from a cDNA clone (Origene ) ( ). .. A carboxy terminal c-Myc tag was added using oligonucleotides (forward: 5′-CACACACGCTAGCCCTCTTAAAAGATCCAAAAAATGAGACTTCTAGC-3′; reverse: 5′-TGTGTGCTCGAGTTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGG AGACTTCTTGAGTGAGTGAATTCCGTG-3′) and subcloned into pAAV8-CAG-MCS (Vector Biolabs) within restriction sites XhoI to NheI to generate pAAV8-CAG-CRRY-Myc (CRRY-Myc) ( ).

Mutagenesis:

Article Title: Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils *
Article Snippet: .. Cloning and Expression of Human MBOAT Proteins in the Yeast ale1 Δ Mutant —Plasmids containing cDNA coding for human MBOAT1 (GenBank™ accession number NM_ 001080480.1), MBOAT2 (GenBank accession number ), and MBOAT5 (GenBank accession number ) were purchased from Origene (Rockville, MD), and a cDNA clone for MBOAT7 (GenBank accession number ) was purchased from Open Biosystems (Huntsville, AL). .. The open reading frames were subcloned into the pYES2.1-TOPO vector according to the manufacturer's instructions.

Real-time Polymerase Chain Reaction:

Article Title: The protein kinase Pak4 disrupts mammary acinar architecture and promotes mammary tumorigenesis
Article Snippet: .. Real time PCR quantitative measurements of Pak4 mRNA were made on cDNA samples provided by OriGene. .. Briefly, primers to human Pak4 were designed using ABI Prism Primer Express software (Applied Biosystems, Foster City, CA).

Expressing:

Article Title: Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils *
Article Snippet: .. Cloning and Expression of Human MBOAT Proteins in the Yeast ale1 Δ Mutant —Plasmids containing cDNA coding for human MBOAT1 (GenBank™ accession number NM_ 001080480.1), MBOAT2 (GenBank accession number ), and MBOAT5 (GenBank accession number ) were purchased from Origene (Rockville, MD), and a cDNA clone for MBOAT7 (GenBank accession number ) was purchased from Open Biosystems (Huntsville, AL). .. The open reading frames were subcloned into the pYES2.1-TOPO vector according to the manufacturer's instructions.

Polymerase Chain Reaction:

Article Title: Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease
Article Snippet: .. The murine CRRY gene was amplified by PCR from a cDNA clone (Origene ) ( ). .. A carboxy terminal c-Myc tag was added using oligonucleotides (forward: 5′-CACACACGCTAGCCCTCTTAAAAGATCCAAAAAATGAGACTTCTAGC-3′; reverse: 5′-TGTGTGCTCGAGTTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGG AGACTTCTTGAGTGAGTGAATTCCGTG-3′) and subcloned into pAAV8-CAG-MCS (Vector Biolabs) within restriction sites XhoI to NheI to generate pAAV8-CAG-CRRY-Myc (CRRY-Myc) ( ).

Over Expression:

Article Title: Cellular Functions of the Autism Risk Factor PTCHD1 in Mice
Article Snippet: .. The pEGFP-C1-Ptchd1 vector used for overexpression assays was created with the cDNA from OriGene. .. P4 Ptchd1 −/y males and littermate wild-type (WT) controls were weighed and then injected intraperitoneally with 100 mg/kg BrdU.

Plasmid Preparation:

Article Title: Cellular Functions of the Autism Risk Factor PTCHD1 in Mice
Article Snippet: .. The pEGFP-C1-Ptchd1 vector used for overexpression assays was created with the cDNA from OriGene. .. P4 Ptchd1 −/y males and littermate wild-type (WT) controls were weighed and then injected intraperitoneally with 100 mg/kg BrdU.

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  • 88
    OriGene colon cancer cdna array
    BMP4 is required for DJ-1-induced and Wnt signaling-mediated <t>CRC</t> migration and invasion. a Western blot analysis of BMP4 expression in HCT116, SW480, and SW620 cells stable transfected with DJ-1 <t>cDNA,</t> DJ-1 shRNA or vector. b Western blot analysis of BMP4 in HCT116-DJ-1, SW480-DJ-1, and SW620 cells treated with or without 100 μM Sulindac for 36 h. c – e HCT116-DJ-1, SW480-DJ-1, and SW620 cells were transfected with two specific BMP4 siRNAs, respectively. Then, protein level of BMP4 was determined by Western blot ( c ); migration and invasion activity were assessed by wound-healing migration assay ( d , scale bar, 500 μm), transwell migration and matrigel invasion assays ( e , scale bar, 100 μm). f – h SW480, DJ-1-konocdown HCT116 (HCT116-shDJ-1), and SW620 (SW620-shDJ-1) cells were transfected with BMP4 cDNA. Then, BMP4 was examined by Western blot ( f ); migration and invasion activity were assessed by wound-healing migration assay ( g , scale bar, 500 μm), transwell migration and matrigel invasion assays ( h , scale bar, 100 μm). Migration was analyzed at 24 h, invasion at 48 h. * P
    Colon Cancer Cdna Array, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene rat brain cdna library
    Reversal of the 14-3-3θ suppression of <t>rEag1</t> K + currents by the 14-3-3 antagonist difopein. ( A ) GST pull-down assay of the cell lysates prepared from HEK293T cells over-expressing the YFP vector, YFP-difopein, or YFP-R18 mutant. Pull-down products were detected by immunoblotting with the anti-14-3-3θ antibody. Compared to the vector control ( lane 1 ), introduction of difopein ( lane 2 ) resulted in a 75% and 64% reduction in the amount of 14-3-3θ pull-down by the GST-C0 and GST-N207 fusion proteins, respectively. In contrast, no significant difference was observed in the presence of the inactive mutant control ( lane 3 ). ( B ) Normalized mean K + current density recorded from HEK293 cells stably expressing rEag1 channels. As indicated, these stable cell lines were subject to transient transfection with various <t>cDNA</t> constructs. The mean current density at +40 mV for each co-expression condition was normalized with respect to that of the co-expression of rEag1 and difopein. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1-difopein co-expression control (*, t -test: p
    Rat Brain Cdna Library, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat brain cdna library/product/OriGene
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    84
    OriGene mouse nup153 cdna
    Subnuclear position of c-FOS locus with respect to nuclear periphery in HeLa cells. Related to Figure 7 . Distribution of c-FOS locus distance to nuclear periphery in control and <t>NUP153</t> KD HeLa cells was measured based on DNA FISH at the indicated time points +/− EGF (50ng/ml). Frequencies at a normalized distance (ND) of 0.0-0.5 are shown. ND= c-FOS locus to periphery distance/cell diameter (d), where d=(2xnuclear area/π) 0.5 . Control HeLa cells (-EGF, n=182; 15 min EGF, n=186; 30 min EGF, n=150; 60 min EGF, n=146; 90 min EGF, n=181; 120 min EGF, n=139); NUP153 KD HeLa cells (-EGF, n=66; 15 min EGF, n=138; 30 min EGF, n=170; 60 min EGF, n=106; 90 min EGF, n=237; 120 min EGF, n=170). Experiment was repeated twice.
    Mouse Nup153 Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    OriGene human cdna panel
    <t>RIF1-L</t> promotes resistance to Aphidicolin treatment but RIF1-S cannot (A) Illustration of RIF1 exon structure and construction of HCT116 mAC-RIF1-L and mAC-RIF1-S cell lines. Constructs containing <t>cDNA</t> encoding the C-terminal portion of either RIF1-L or RIF1-S were inserted at the end of exon 29, by CRISPR-mediated integration of donor plasmids into the HCT116 mAC-RIF1 cell line. (B) Expression of RIF1 in HCT116 mAC-RIF1, mAC-RIF1-L, mAC-RIF1-S and RIF1 KO cell lines. Whole cell extracts were harvested for western blotting with anti-RIF1 antibody. Loading control shows tubulin. (C) CFA comparing resistance to Aphidicolin of mAC-RIF1(black bars), mAC-RIF1-L (red bars), mAC-RIF1-S (grey bars), and RIF1-KO (open bars) cell lines. *p
    Human Cdna Panel, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BMP4 is required for DJ-1-induced and Wnt signaling-mediated CRC migration and invasion. a Western blot analysis of BMP4 expression in HCT116, SW480, and SW620 cells stable transfected with DJ-1 cDNA, DJ-1 shRNA or vector. b Western blot analysis of BMP4 in HCT116-DJ-1, SW480-DJ-1, and SW620 cells treated with or without 100 μM Sulindac for 36 h. c – e HCT116-DJ-1, SW480-DJ-1, and SW620 cells were transfected with two specific BMP4 siRNAs, respectively. Then, protein level of BMP4 was determined by Western blot ( c ); migration and invasion activity were assessed by wound-healing migration assay ( d , scale bar, 500 μm), transwell migration and matrigel invasion assays ( e , scale bar, 100 μm). f – h SW480, DJ-1-konocdown HCT116 (HCT116-shDJ-1), and SW620 (SW620-shDJ-1) cells were transfected with BMP4 cDNA. Then, BMP4 was examined by Western blot ( f ); migration and invasion activity were assessed by wound-healing migration assay ( g , scale bar, 500 μm), transwell migration and matrigel invasion assays ( h , scale bar, 100 μm). Migration was analyzed at 24 h, invasion at 48 h. * P

    Journal: Cell Death & Disease

    Article Title: DJ-1 promotes colorectal cancer progression through activating PLAGL2/Wnt/BMP4 axis

    doi: 10.1038/s41419-018-0883-4

    Figure Lengend Snippet: BMP4 is required for DJ-1-induced and Wnt signaling-mediated CRC migration and invasion. a Western blot analysis of BMP4 expression in HCT116, SW480, and SW620 cells stable transfected with DJ-1 cDNA, DJ-1 shRNA or vector. b Western blot analysis of BMP4 in HCT116-DJ-1, SW480-DJ-1, and SW620 cells treated with or without 100 μM Sulindac for 36 h. c – e HCT116-DJ-1, SW480-DJ-1, and SW620 cells were transfected with two specific BMP4 siRNAs, respectively. Then, protein level of BMP4 was determined by Western blot ( c ); migration and invasion activity were assessed by wound-healing migration assay ( d , scale bar, 500 μm), transwell migration and matrigel invasion assays ( e , scale bar, 100 μm). f – h SW480, DJ-1-konocdown HCT116 (HCT116-shDJ-1), and SW620 (SW620-shDJ-1) cells were transfected with BMP4 cDNA. Then, BMP4 was examined by Western blot ( f ); migration and invasion activity were assessed by wound-healing migration assay ( g , scale bar, 500 μm), transwell migration and matrigel invasion assays ( h , scale bar, 100 μm). Migration was analyzed at 24 h, invasion at 48 h. * P

    Article Snippet: Consistently, elevated DJ-1 mRNA was also observed in CRC tissues using qRT-PCR in an OriGene Colon Cancer cDNA Array (Fig. , P = 0.016).

    Techniques: Migration, Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Activity Assay

    DJ-1 activates Wnt signaling by enhancing PLAGL2 expression. a Western blot analysis of PLAGL2 expression in HCT116, SW480, and SW620 cells stably transfected with DJ-1 cDNA, DJ-1 shRNA, or vector. b – f HCT116-DJ-1, SW480-DJ-1, and SW620 cells were transfected with two specific PLAGL2 (PL) siRNAs, respectively. Then, protein levels of activated-β-catenin, BMP4, CCND1, and PLAGL2 were determined by Western blot ( b ); Wnt activity was analyzed by TOP-Flash/FOP-Flash assay ( c ); proliferation of CRC cells was examined by CCK8 kit ( d ); proliferative activity was detected by colony formation assay ( e ); migration and invasion activity were assessed by transwell migration and matrigel invasion assays ( f , scale bar, 100 μm). Migration was analyzed at 24 h, invasion at 48 h. * P

    Journal: Cell Death & Disease

    Article Title: DJ-1 promotes colorectal cancer progression through activating PLAGL2/Wnt/BMP4 axis

    doi: 10.1038/s41419-018-0883-4

    Figure Lengend Snippet: DJ-1 activates Wnt signaling by enhancing PLAGL2 expression. a Western blot analysis of PLAGL2 expression in HCT116, SW480, and SW620 cells stably transfected with DJ-1 cDNA, DJ-1 shRNA, or vector. b – f HCT116-DJ-1, SW480-DJ-1, and SW620 cells were transfected with two specific PLAGL2 (PL) siRNAs, respectively. Then, protein levels of activated-β-catenin, BMP4, CCND1, and PLAGL2 were determined by Western blot ( b ); Wnt activity was analyzed by TOP-Flash/FOP-Flash assay ( c ); proliferation of CRC cells was examined by CCK8 kit ( d ); proliferative activity was detected by colony formation assay ( e ); migration and invasion activity were assessed by transwell migration and matrigel invasion assays ( f , scale bar, 100 μm). Migration was analyzed at 24 h, invasion at 48 h. * P

    Article Snippet: Consistently, elevated DJ-1 mRNA was also observed in CRC tissues using qRT-PCR in an OriGene Colon Cancer cDNA Array (Fig. , P = 0.016).

    Techniques: Expressing, Western Blot, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Activity Assay, Colony Assay, Migration

    DJ-1 is upregulated in human colorectal cancers and associated with CRC progression. a qRT - PCR was conducted on CRC samples in the Tissue Scan Colon Cancer cDNA Array V to determine DJ-1 mRNA expression. b Immunohistochemical staining of DJ-1 in paraffin-embedded human CRC tissues. c Immunohistochemical scores for DJ-1 in normal colorectal mucosa and CRC tissues. d Immunohistochemical staining of DJ-1 in mucosa plus submucosa, muscle, and serosa tissues, which exhibit different invasion depth (left). The relative intensity of DJ-1 in different tumor size (≤3 or > 3 cm) (right). e Immunohistochemical staining of DJ-1 in well, moderately, and poorly differentiated CRC tissues. f Immunohistochemical staining of DJ-1 in different stage (I, II, III/IV) CRC tissues. g Kaplan–Meier survival curves of CRC patients with low ( n = 47) and high ( n = 60) DJ-1 expression. * P

    Journal: Cell Death & Disease

    Article Title: DJ-1 promotes colorectal cancer progression through activating PLAGL2/Wnt/BMP4 axis

    doi: 10.1038/s41419-018-0883-4

    Figure Lengend Snippet: DJ-1 is upregulated in human colorectal cancers and associated with CRC progression. a qRT - PCR was conducted on CRC samples in the Tissue Scan Colon Cancer cDNA Array V to determine DJ-1 mRNA expression. b Immunohistochemical staining of DJ-1 in paraffin-embedded human CRC tissues. c Immunohistochemical scores for DJ-1 in normal colorectal mucosa and CRC tissues. d Immunohistochemical staining of DJ-1 in mucosa plus submucosa, muscle, and serosa tissues, which exhibit different invasion depth (left). The relative intensity of DJ-1 in different tumor size (≤3 or > 3 cm) (right). e Immunohistochemical staining of DJ-1 in well, moderately, and poorly differentiated CRC tissues. f Immunohistochemical staining of DJ-1 in different stage (I, II, III/IV) CRC tissues. g Kaplan–Meier survival curves of CRC patients with low ( n = 47) and high ( n = 60) DJ-1 expression. * P

    Article Snippet: Consistently, elevated DJ-1 mRNA was also observed in CRC tissues using qRT-PCR in an OriGene Colon Cancer cDNA Array (Fig. , P = 0.016).

    Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining

    DJ-1 enhances CRC tumor growth and metastasis in vivo. HCT116 cells were stably transfected with DJ-1 shRNA or negative control vector (NC), or stably transfected with DJ-1 cDNA or vector. a – c Transfected HCT116 cells were subcutaneously injected into nude mice and xenograft tumor growth was determined for 25 days or 45 days. Tumor mass images ( a ). Growth curves of tumor volumes ( b ). Tumor weight ( c ). n = 4. d Immunohistochemical staining of PCNA in xenografts from a . PCNA-positive cells were calculated. Scale bar, 100 μm. e Histopathology showing the lung metastases in mice and quantification of number of metastases following tail-vein injection. n = 6. * P

    Journal: Cell Death & Disease

    Article Title: DJ-1 promotes colorectal cancer progression through activating PLAGL2/Wnt/BMP4 axis

    doi: 10.1038/s41419-018-0883-4

    Figure Lengend Snippet: DJ-1 enhances CRC tumor growth and metastasis in vivo. HCT116 cells were stably transfected with DJ-1 shRNA or negative control vector (NC), or stably transfected with DJ-1 cDNA or vector. a – c Transfected HCT116 cells were subcutaneously injected into nude mice and xenograft tumor growth was determined for 25 days or 45 days. Tumor mass images ( a ). Growth curves of tumor volumes ( b ). Tumor weight ( c ). n = 4. d Immunohistochemical staining of PCNA in xenografts from a . PCNA-positive cells were calculated. Scale bar, 100 μm. e Histopathology showing the lung metastases in mice and quantification of number of metastases following tail-vein injection. n = 6. * P

    Article Snippet: Consistently, elevated DJ-1 mRNA was also observed in CRC tissues using qRT-PCR in an OriGene Colon Cancer cDNA Array (Fig. , P = 0.016).

    Techniques: In Vivo, Stable Transfection, Transfection, shRNA, Negative Control, Plasmid Preparation, Injection, Mouse Assay, Immunohistochemistry, Staining, Histopathology

    Reversal of the 14-3-3θ suppression of rEag1 K + currents by the 14-3-3 antagonist difopein. ( A ) GST pull-down assay of the cell lysates prepared from HEK293T cells over-expressing the YFP vector, YFP-difopein, or YFP-R18 mutant. Pull-down products were detected by immunoblotting with the anti-14-3-3θ antibody. Compared to the vector control ( lane 1 ), introduction of difopein ( lane 2 ) resulted in a 75% and 64% reduction in the amount of 14-3-3θ pull-down by the GST-C0 and GST-N207 fusion proteins, respectively. In contrast, no significant difference was observed in the presence of the inactive mutant control ( lane 3 ). ( B ) Normalized mean K + current density recorded from HEK293 cells stably expressing rEag1 channels. As indicated, these stable cell lines were subject to transient transfection with various cDNA constructs. The mean current density at +40 mV for each co-expression condition was normalized with respect to that of the co-expression of rEag1 and difopein. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1-difopein co-expression control (*, t -test: p

    Journal: PLoS ONE

    Article Title: 14-3-3? is a Binding Partner of Rat Eag1 Potassium Channels

    doi: 10.1371/journal.pone.0041203

    Figure Lengend Snippet: Reversal of the 14-3-3θ suppression of rEag1 K + currents by the 14-3-3 antagonist difopein. ( A ) GST pull-down assay of the cell lysates prepared from HEK293T cells over-expressing the YFP vector, YFP-difopein, or YFP-R18 mutant. Pull-down products were detected by immunoblotting with the anti-14-3-3θ antibody. Compared to the vector control ( lane 1 ), introduction of difopein ( lane 2 ) resulted in a 75% and 64% reduction in the amount of 14-3-3θ pull-down by the GST-C0 and GST-N207 fusion proteins, respectively. In contrast, no significant difference was observed in the presence of the inactive mutant control ( lane 3 ). ( B ) Normalized mean K + current density recorded from HEK293 cells stably expressing rEag1 channels. As indicated, these stable cell lines were subject to transient transfection with various cDNA constructs. The mean current density at +40 mV for each co-expression condition was normalized with respect to that of the co-expression of rEag1 and difopein. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1-difopein co-expression control (*, t -test: p

    Article Snippet: Interaction of 14-3-3θ with the N- and C-termini of rEag1 in vitro We carried out the yeast two-hybrid screening of a rat brain cDNA library by using the N-terminus (amino acids 1–207) of the rEag1 protein (rEag1-N207) as the bait ( ).

    Techniques: Pull Down Assay, Expressing, Plasmid Preparation, Mutagenesis, Stable Transfection, Transfection, Construct

    Interaction of rEag1 N- and C-termini with 14-3-3θ. ( A ) Schematic representation of ( top ) the structural topology of the rEag1 channel and ( bottom ) the rEag1 GST-N207 and GST-C0 fusion proteins. ( B ) Yeast two-hybrid assay. cDNA encoding rEag1-N207 or C0 segment was fused to the coding sequence for LexA DNA binding domain and subcloned into the pGilda vector. cDNA for the B42 transcriptional activation domain alone ( Empty ) or in combination with 14-3-3θ was subcloned into the pJG4-5 vector. Yeasts co-transformed with the pGilda- and the pJG4-5-based plasmids were streaked on leucine-lacking plates. ( C ) GST pull-down assay of in vitro translated 14-3-3θ. Pull-down products were immunoblotted with the anti-14-3-3θ antibody. Indicated to the left are the molecular weight markers (in kDa). ( D,E ) Cell lysates prepared from HEK293T cells expressing myc-14-3-3θ were used for GST pull-down assay with GST or the fusion protein GST-N207/GST-C0. ( Left panels ) Coomassie blue staining of the GST proteins. ( Right panels ) Immunoblotting of pull-down products with the anti-myc antibody. Input volume was 5% of that of the cell lysates for pull-down.

    Journal: PLoS ONE

    Article Title: 14-3-3? is a Binding Partner of Rat Eag1 Potassium Channels

    doi: 10.1371/journal.pone.0041203

    Figure Lengend Snippet: Interaction of rEag1 N- and C-termini with 14-3-3θ. ( A ) Schematic representation of ( top ) the structural topology of the rEag1 channel and ( bottom ) the rEag1 GST-N207 and GST-C0 fusion proteins. ( B ) Yeast two-hybrid assay. cDNA encoding rEag1-N207 or C0 segment was fused to the coding sequence for LexA DNA binding domain and subcloned into the pGilda vector. cDNA for the B42 transcriptional activation domain alone ( Empty ) or in combination with 14-3-3θ was subcloned into the pJG4-5 vector. Yeasts co-transformed with the pGilda- and the pJG4-5-based plasmids were streaked on leucine-lacking plates. ( C ) GST pull-down assay of in vitro translated 14-3-3θ. Pull-down products were immunoblotted with the anti-14-3-3θ antibody. Indicated to the left are the molecular weight markers (in kDa). ( D,E ) Cell lysates prepared from HEK293T cells expressing myc-14-3-3θ were used for GST pull-down assay with GST or the fusion protein GST-N207/GST-C0. ( Left panels ) Coomassie blue staining of the GST proteins. ( Right panels ) Immunoblotting of pull-down products with the anti-myc antibody. Input volume was 5% of that of the cell lysates for pull-down.

    Article Snippet: Interaction of 14-3-3θ with the N- and C-termini of rEag1 in vitro We carried out the yeast two-hybrid screening of a rat brain cDNA library by using the N-terminus (amino acids 1–207) of the rEag1 protein (rEag1-N207) as the bait ( ).

    Techniques: Y2H Assay, Sequencing, Binding Assay, Plasmid Preparation, Activation Assay, Transformation Assay, Pull Down Assay, In Vitro, Molecular Weight, Expressing, Staining

    Subnuclear position of c-FOS locus with respect to nuclear periphery in HeLa cells. Related to Figure 7 . Distribution of c-FOS locus distance to nuclear periphery in control and NUP153 KD HeLa cells was measured based on DNA FISH at the indicated time points +/− EGF (50ng/ml). Frequencies at a normalized distance (ND) of 0.0-0.5 are shown. ND= c-FOS locus to periphery distance/cell diameter (d), where d=(2xnuclear area/π) 0.5 . Control HeLa cells (-EGF, n=182; 15 min EGF, n=186; 30 min EGF, n=150; 60 min EGF, n=146; 90 min EGF, n=181; 120 min EGF, n=139); NUP153 KD HeLa cells (-EGF, n=66; 15 min EGF, n=138; 30 min EGF, n=170; 60 min EGF, n=106; 90 min EGF, n=237; 120 min EGF, n=170). Experiment was repeated twice.

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: Subnuclear position of c-FOS locus with respect to nuclear periphery in HeLa cells. Related to Figure 7 . Distribution of c-FOS locus distance to nuclear periphery in control and NUP153 KD HeLa cells was measured based on DNA FISH at the indicated time points +/− EGF (50ng/ml). Frequencies at a normalized distance (ND) of 0.0-0.5 are shown. ND= c-FOS locus to periphery distance/cell diameter (d), where d=(2xnuclear area/π) 0.5 . Control HeLa cells (-EGF, n=182; 15 min EGF, n=186; 30 min EGF, n=150; 60 min EGF, n=146; 90 min EGF, n=181; 120 min EGF, n=139); NUP153 KD HeLa cells (-EGF, n=66; 15 min EGF, n=138; 30 min EGF, n=170; 60 min EGF, n=106; 90 min EGF, n=237; 120 min EGF, n=170). Experiment was repeated twice.

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Fluorescence In Situ Hybridization

    NUP153 influences transcription and binding of CTCF and cohesin at bivalent genes. (A) Scatter plot showing transcript expression levels in Log2[CPM] scale in control and NUP153 KD-1 ES cells. Blue points are all differentially expressed genes (n=711) and orange points are differentially expressed bivalent genes. (B) Table showing number of differentially expressed genes that associate with all, Group I and Group II CTCF-positive TSS (top). Plots showing number of differentially regulated NUP153-positive and NUP153-negative genes that associate with Group I and II CTCF-positive TSS (bottom). (C) NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq, and transcripts (RNA-Seq) tracks are shown for two NUP153-positive Group I genes, Rtn4rl1 (left panel) and Calb2 (right panel) in control (WT) and NUP153 KD ES cells. Rtn4rl1 shows transcriptional up regulation and Calb2 shown transcriptional down regulation. (D) NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq tracks are shown for a 145-150 kb region for HoxA and HoxC loci in control (WT) and NUP153 KD ES cells as indicated. Arrows point to regions where CTCF or SMC3 binding are altered in NUP153 KD ES cells. CTCF sites labeled with asterisk (*) point to CTCF sites that have been reported to regulate transcription at Hox loci by mediating formation of TADs 71 , 75 . The 2D heat map shows the interaction frequency in mouse ES cells 61 . Hi-C data was aligned to the mm9 genome showing HoxA cluster residing in a TAD boundary and HoxC cluster in a TAD as published 61 . H3K4me3 and H3K27me3 57 and CBP/P300 60 ChIP-Seq data were previously published. CPM, Counts per million.

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: NUP153 influences transcription and binding of CTCF and cohesin at bivalent genes. (A) Scatter plot showing transcript expression levels in Log2[CPM] scale in control and NUP153 KD-1 ES cells. Blue points are all differentially expressed genes (n=711) and orange points are differentially expressed bivalent genes. (B) Table showing number of differentially expressed genes that associate with all, Group I and Group II CTCF-positive TSS (top). Plots showing number of differentially regulated NUP153-positive and NUP153-negative genes that associate with Group I and II CTCF-positive TSS (bottom). (C) NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq, and transcripts (RNA-Seq) tracks are shown for two NUP153-positive Group I genes, Rtn4rl1 (left panel) and Calb2 (right panel) in control (WT) and NUP153 KD ES cells. Rtn4rl1 shows transcriptional up regulation and Calb2 shown transcriptional down regulation. (D) NUP153 DamID-Seq, CTCF, cohesin, H3K4me3, and H3K27me3 ChIP-Seq tracks are shown for a 145-150 kb region for HoxA and HoxC loci in control (WT) and NUP153 KD ES cells as indicated. Arrows point to regions where CTCF or SMC3 binding are altered in NUP153 KD ES cells. CTCF sites labeled with asterisk (*) point to CTCF sites that have been reported to regulate transcription at Hox loci by mediating formation of TADs 71 , 75 . The 2D heat map shows the interaction frequency in mouse ES cells 61 . Hi-C data was aligned to the mm9 genome showing HoxA cluster residing in a TAD boundary and HoxC cluster in a TAD as published 61 . H3K4me3 and H3K27me3 57 and CBP/P300 60 ChIP-Seq data were previously published. CPM, Counts per million.

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay, Labeling, Hi-C

    IEG loci are NUP153 targets in mouse ES cells. Related to Figure 3 . CBP/P300, H3K4me3, H3K27me3, NUP153, CTCF, SMC3 ChIP-Seq, NUP153 DamID-Seq and RNA-Seq tracks are shown for IEG loci, Egr1 (top panel), and Jun (bottom panel) in control and NUP153 KD ES cells. CBP/P300 60 , H3K4me3, H3K27me3 ChIP-Seq data 57 were previously published

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: IEG loci are NUP153 targets in mouse ES cells. Related to Figure 3 . CBP/P300, H3K4me3, H3K27me3, NUP153, CTCF, SMC3 ChIP-Seq, NUP153 DamID-Seq and RNA-Seq tracks are shown for IEG loci, Egr1 (top panel), and Jun (bottom panel) in control and NUP153 KD ES cells. CBP/P300 60 , H3K4me3, H3K27me3 ChIP-Seq data 57 were previously published

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Chromatin Immunoprecipitation, RNA Sequencing Assay

    Distribution of NUP153, CTCF, SMC3 and histone modifications across genetic elements in control (WT) mouse ES cells, and characteristics of NUP153 deficient mouse ES cells. Related to Figure 2 and 3 . (A) Metagene profiles showing mean CTCF, SMC3 and NUP153 binding at TSS (+/-5kb) (n=24,352). (B) Metagene profiles showing mean CTCF, SMC3 and NUP153 binding +/− 5kb of transcriptionally active (n=8,768) and inactive (n11,861) TSS. (C) Metagene profiles showing mean NUP153, H3K27Ac 59 , H3K4me1 58 , CBP/P300 60 , CTCF, and SMC3 binding at enhancers (n=16,242)(+/-5kb). (D) Metagene profiles showing distribution of H3K27Ac 59 , H3K4me1 58 , CBP/P300 60 at NUP153-positive (n=2,849) and NUP153-negative (n=13,393) enhancers (+/− 2.5kb) in control ES cells. (E) Distribution of CTCF or cohesin binding sites was evaluated to determine the median of their distance to the nearest NUP153 binding sites in mouse ES cells. CTCF and cohesin binding sites exhibit a median of ∼5 kb distance to the nearest NUP153 binding sites. (F) Real time RT-PCR showing relative NUP153 mRNA levels in control (scramble) and NUP153 shRNA (KD-1, KD-2) virus transduced ES cells. GAPDH mRNA level was used to normalize mRNA levels. Student’s t-test was applied to calculate significance. Mean mRNA levels ± standard error mean. ** p

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: Distribution of NUP153, CTCF, SMC3 and histone modifications across genetic elements in control (WT) mouse ES cells, and characteristics of NUP153 deficient mouse ES cells. Related to Figure 2 and 3 . (A) Metagene profiles showing mean CTCF, SMC3 and NUP153 binding at TSS (+/-5kb) (n=24,352). (B) Metagene profiles showing mean CTCF, SMC3 and NUP153 binding +/− 5kb of transcriptionally active (n=8,768) and inactive (n11,861) TSS. (C) Metagene profiles showing mean NUP153, H3K27Ac 59 , H3K4me1 58 , CBP/P300 60 , CTCF, and SMC3 binding at enhancers (n=16,242)(+/-5kb). (D) Metagene profiles showing distribution of H3K27Ac 59 , H3K4me1 58 , CBP/P300 60 at NUP153-positive (n=2,849) and NUP153-negative (n=13,393) enhancers (+/− 2.5kb) in control ES cells. (E) Distribution of CTCF or cohesin binding sites was evaluated to determine the median of their distance to the nearest NUP153 binding sites in mouse ES cells. CTCF and cohesin binding sites exhibit a median of ∼5 kb distance to the nearest NUP153 binding sites. (F) Real time RT-PCR showing relative NUP153 mRNA levels in control (scramble) and NUP153 shRNA (KD-1, KD-2) virus transduced ES cells. GAPDH mRNA level was used to normalize mRNA levels. Student’s t-test was applied to calculate significance. Mean mRNA levels ± standard error mean. ** p

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Binding Assay, Quantitative RT-PCR, shRNA

    Transcription and chromatin structure at IEG loci in HeLa cells. Related to Figure 4 and 5 . (A) Real-time RT-PCR showing relative mRNA and nascent mRNA levels for c-FOS gene in control and NUP153 KD HeLa cells in a time course dependent manner. GAPDH was used to normalize mRNA levels. Values are mean ± standard deviation. Student’s t-test was applied to calculate significance. * p

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: Transcription and chromatin structure at IEG loci in HeLa cells. Related to Figure 4 and 5 . (A) Real-time RT-PCR showing relative mRNA and nascent mRNA levels for c-FOS gene in control and NUP153 KD HeLa cells in a time course dependent manner. GAPDH was used to normalize mRNA levels. Values are mean ± standard deviation. Student’s t-test was applied to calculate significance. * p

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Quantitative RT-PCR, Standard Deviation

    IEGs associate with the NPC in NUP153-dependent manner during paused state and transcription initiation. (A) Immunostaining of Lamin B1 and c-FOS DNA FISH in control and NUP153 KD HeLa cells are shown at the indicated time points. Cell numbers are as indicated. HeLa cells contain three c-FOS alleles (white arrows). Scale bar, 5μm. (B) Cumulative frequency graphs showing distribution of the c-FOS locus distance to nuclear periphery in control and NUP153 KD HeLa cells at the indicated time points. Cumulative frequencies at a normalized distance (ND) of 0.0-0.12 are shown. See Figure S7 for distribution of loci in all cells that were analyzed (ND of 0.0-0.5). ND= c-FOS locus to periphery distance/cell diameter (d), where d=(2xnuclear area/ π ) 0.5 . * p

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: IEGs associate with the NPC in NUP153-dependent manner during paused state and transcription initiation. (A) Immunostaining of Lamin B1 and c-FOS DNA FISH in control and NUP153 KD HeLa cells are shown at the indicated time points. Cell numbers are as indicated. HeLa cells contain three c-FOS alleles (white arrows). Scale bar, 5μm. (B) Cumulative frequency graphs showing distribution of the c-FOS locus distance to nuclear periphery in control and NUP153 KD HeLa cells at the indicated time points. Cumulative frequencies at a normalized distance (ND) of 0.0-0.12 are shown. See Figure S7 for distribution of loci in all cells that were analyzed (ND of 0.0-0.5). ND= c-FOS locus to periphery distance/cell diameter (d), where d=(2xnuclear area/ π ) 0.5 . * p

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Immunostaining, Fluorescence In Situ Hybridization

    NUP153 interacts with CTCF and cohesin. (A) Silver stain showing proteins that IP with FLAG-NUP153. (B) Co-IP shows FLAG-NUP153 interaction with CTCF, and cohesin subunits, SMC3, SMC1A, RAD21. NUP153 was pulled down using anti-FLAG antibody (Ab). (C) Schematic showing steps of chromatin fractionation assay in HeLa cells. (D) NUP153 was detected in the nuclear insoluble fraction (P2) along with CTCF and cohesin. NUP153 detected in the chromatin-associated soluble fraction (S3) following micrococcal nuclease (MNase) treatment of P1 fraction. Ppt, precipitate; Sup, supernatant; Nucleoporin 62, NUP62; Loading controls: α -Tubulin (cytoplasm), Histone H3 (chromatin).

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: NUP153 interacts with CTCF and cohesin. (A) Silver stain showing proteins that IP with FLAG-NUP153. (B) Co-IP shows FLAG-NUP153 interaction with CTCF, and cohesin subunits, SMC3, SMC1A, RAD21. NUP153 was pulled down using anti-FLAG antibody (Ab). (C) Schematic showing steps of chromatin fractionation assay in HeLa cells. (D) NUP153 was detected in the nuclear insoluble fraction (P2) along with CTCF and cohesin. NUP153 detected in the chromatin-associated soluble fraction (S3) following micrococcal nuclease (MNase) treatment of P1 fraction. Ppt, precipitate; Sup, supernatant; Nucleoporin 62, NUP62; Loading controls: α -Tubulin (cytoplasm), Histone H3 (chromatin).

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Silver Staining, Co-Immunoprecipitation Assay, Fractionation

    Distribution of CTCF, SMC3 and NUP153 across genetic elements and CTCF-positive sites in mouse ES cells. Related to Figure 2 and 3 . (A) Distribution of CTCF and SMC3 sites across the indicated genetic elements in control and NUP153 KD-1 and KD-2 mouse ES cells. (B) Metagene profiles showing mean NUP153 binding at CTCF-positive TSS, enhancer and TAD boundaries. (C) Table showing mean CTCF binding at CTCF-positive TSS, enhancer and TAD boundaries in control and NUP153 KD ES cells. (D-E) Metagene profiles showing mean CTCF (D) and SMC3 (E) binding in control versus NUP153 KD cells at CTCF-positive Group I and Group II TSS, enhancer and TAD boundaries. Number of CTCF-positive sites for each Group is as indicated.

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: Distribution of CTCF, SMC3 and NUP153 across genetic elements and CTCF-positive sites in mouse ES cells. Related to Figure 2 and 3 . (A) Distribution of CTCF and SMC3 sites across the indicated genetic elements in control and NUP153 KD-1 and KD-2 mouse ES cells. (B) Metagene profiles showing mean NUP153 binding at CTCF-positive TSS, enhancer and TAD boundaries. (C) Table showing mean CTCF binding at CTCF-positive TSS, enhancer and TAD boundaries in control and NUP153 KD ES cells. (D-E) Metagene profiles showing mean CTCF (D) and SMC3 (E) binding in control versus NUP153 KD cells at CTCF-positive Group I and Group II TSS, enhancer and TAD boundaries. Number of CTCF-positive sites for each Group is as indicated.

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Binding Assay

    NUP153 controls POL II recruitment to IEG promoters and impact IEG transcription initiation. (A) Western blot showing NUP153 protein levels in HeLa cells transduced with control or NUP153 shRNA (KD-1 and KD-2) virus. (B) Real-time RT-PCR showing relative IEG mRNA and IEG nascent mRNA levels in control and NUP153 KD HeLa cells in a time course dependent manner. (C) Real-time RT-PCR showing relative IEG mRNA levels in control, NUP153 KD and NUP153 KD cells that express FLAG-NUP153 HeLa cells upon 15 min EGF treatment. GAPDH was used to normalize mRNA levels. (D) POL II binding across the IEGs, JUN and EGR1 loci, was mapped by POL II ChIP in control and NUP153 KD HeLa cells at the paused state (minus EGF) and upon transcription induction 15 min and 30 min of EGF treatment. POL II occupancy at various IEG genetic elements (promoter, TSS, gene body (GB), and transcription termination site (TTS)) was measured using real-time PCR primers (see Table S1 ) as denoted in the schematics showing EGR1 and JUN genes. Data shown are percent (%) of input at each genetic element. Values are mean ± standard deviation. Student’s t-test was applied to calculate significance. * p

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: NUP153 controls POL II recruitment to IEG promoters and impact IEG transcription initiation. (A) Western blot showing NUP153 protein levels in HeLa cells transduced with control or NUP153 shRNA (KD-1 and KD-2) virus. (B) Real-time RT-PCR showing relative IEG mRNA and IEG nascent mRNA levels in control and NUP153 KD HeLa cells in a time course dependent manner. (C) Real-time RT-PCR showing relative IEG mRNA levels in control, NUP153 KD and NUP153 KD cells that express FLAG-NUP153 HeLa cells upon 15 min EGF treatment. GAPDH was used to normalize mRNA levels. (D) POL II binding across the IEGs, JUN and EGR1 loci, was mapped by POL II ChIP in control and NUP153 KD HeLa cells at the paused state (minus EGF) and upon transcription induction 15 min and 30 min of EGF treatment. POL II occupancy at various IEG genetic elements (promoter, TSS, gene body (GB), and transcription termination site (TTS)) was measured using real-time PCR primers (see Table S1 ) as denoted in the schematics showing EGR1 and JUN genes. Data shown are percent (%) of input at each genetic element. Values are mean ± standard deviation. Student’s t-test was applied to calculate significance. * p

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Western Blot, Transduction, shRNA, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

    Analyses of NUP153 deficient HeLa cells for nucleocytoplasmic trafficking. Related to Figure 4 . (A) Nuclear import and export were tracked using the dexamethasone responsive REV-GFP-GR construct (see Methods for more detailed information). Shown are representative images of control and NUP153 KD HeLa cells after dexamethasone (Dex) treatment or wash off at the indicated time points. To evaluate GR import, cells were treated with 250 mM Dex at the indicated times (n=68-84). To evaluate GR export, Dex was washed off after 120 mins (considered zero (0) time point) (n=75). (B) Graphs showing nuclear import of REV-GFP-GR after Dex treatment (left) and export of REV-GFP-GR after Dex wash off (right). Values were calculated based on % of cells which show nuclear GR-GFP signal after Dex treatment, or Dex wash off at the indicated time points. Scale bar, 10μm. (C) Oligo(dT)50-Cy3 RNA FISH in control and NUP153 KD HeLa cells was performed to evaluate Poly(A) + RNA export. Scale 10μm.

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: Analyses of NUP153 deficient HeLa cells for nucleocytoplasmic trafficking. Related to Figure 4 . (A) Nuclear import and export were tracked using the dexamethasone responsive REV-GFP-GR construct (see Methods for more detailed information). Shown are representative images of control and NUP153 KD HeLa cells after dexamethasone (Dex) treatment or wash off at the indicated time points. To evaluate GR import, cells were treated with 250 mM Dex at the indicated times (n=68-84). To evaluate GR export, Dex was washed off after 120 mins (considered zero (0) time point) (n=75). (B) Graphs showing nuclear import of REV-GFP-GR after Dex treatment (left) and export of REV-GFP-GR after Dex wash off (right). Values were calculated based on % of cells which show nuclear GR-GFP signal after Dex treatment, or Dex wash off at the indicated time points. Scale bar, 10μm. (C) Oligo(dT)50-Cy3 RNA FISH in control and NUP153 KD HeLa cells was performed to evaluate Poly(A) + RNA export. Scale 10μm.

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Construct, Fluorescence In Situ Hybridization

    FLAG-NUP153 expression in HEK293T cells and generation of NUP153-Dam mouse ES cell lines. Related to Figure 1 , 2 , 3 . (A) Western blot showing validation of FLAG-NUP153 expression. Whole cell extracts were prepared from FLAG-GFP- or FLAG-NUP153-expressing HEK293T cells and were subjected to western blot using anti-NUP153, and anti-FLAG antibodies as indicated. ACTIN was used as an internal control. (B) Cellular localization of FLAG-NUP153 protein in HEK293T cells was determined by immunostaining. Scale, 10μm. (C) Mouse NUP153-cDNA (4.5 kb) (ATCC) was cloned into Kpn I and Xho I sites in pIND-(V5)-EcoDam plasmid 56 to generate NUP153-Dam plasmid. Female mouse ES cell line 16.7 were electroporated using NUP153-Dam or Dam only (pIND-(V5)-EcoDam) plasmids. ES clones were screened by genomic DNA (gDNA) PCR using a primer pair that amplifies a 0.4 kb fragment across V5-tag and Dam sequences. (D) Several ES cell clones were screened for Dam activity by gDNA PCR as previously described 56 . (E) Expression of NUP153-Dam fusion protein was determined by performing immunofluorescence using anti-V5 antibody in Dam only and NUP153-Dam mouse ES cells. Scale, 10μm.

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: FLAG-NUP153 expression in HEK293T cells and generation of NUP153-Dam mouse ES cell lines. Related to Figure 1 , 2 , 3 . (A) Western blot showing validation of FLAG-NUP153 expression. Whole cell extracts were prepared from FLAG-GFP- or FLAG-NUP153-expressing HEK293T cells and were subjected to western blot using anti-NUP153, and anti-FLAG antibodies as indicated. ACTIN was used as an internal control. (B) Cellular localization of FLAG-NUP153 protein in HEK293T cells was determined by immunostaining. Scale, 10μm. (C) Mouse NUP153-cDNA (4.5 kb) (ATCC) was cloned into Kpn I and Xho I sites in pIND-(V5)-EcoDam plasmid 56 to generate NUP153-Dam plasmid. Female mouse ES cell line 16.7 were electroporated using NUP153-Dam or Dam only (pIND-(V5)-EcoDam) plasmids. ES clones were screened by genomic DNA (gDNA) PCR using a primer pair that amplifies a 0.4 kb fragment across V5-tag and Dam sequences. (D) Several ES cell clones were screened for Dam activity by gDNA PCR as previously described 56 . (E) Expression of NUP153-Dam fusion protein was determined by performing immunofluorescence using anti-V5 antibody in Dam only and NUP153-Dam mouse ES cells. Scale, 10μm.

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Expressing, Western Blot, Immunostaining, Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Activity Assay, Immunofluorescence

    NUP153 and CTCF co-regulate POL II recruitment at the IEG paused state. (A) Real-time RT-PCR showing relative CTCF mRNA levels in HeLa cells transfected with control or CTCF shRNA expression vectors (KD-1 and KD-2). (B) Real-time RT-PCR showing relative JUN and EGR1 mRNA levels in control or CTCF KD HeLa cells. (C) POL II binding across the IEGs, JUN and EGR1 loci, was mapped by POL II ChIP in control and CTCF KD HeLa cells at the paused state (minus EGF). POL II occupancy at the indicated genetic elements was measured using real-time PCR primers as denoted in the schematics showing EGR1 and JUN genes ( Table S1 ). Data shown are percent (%) of input at each genetic element. (D) Real-time RT-PCR showing relative EGR1 , JUN , NUP153 and CTCF mRNA levels were measured in NUP153 KD, CTCF KD and in CTCF/NUP153 KD HeLa cells. Values are mean ± standard deviation. Relative mRNA levels were normalized using GAPDH mRNA levels. Student’s t-test was applied to calculate significance. * p

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: NUP153 and CTCF co-regulate POL II recruitment at the IEG paused state. (A) Real-time RT-PCR showing relative CTCF mRNA levels in HeLa cells transfected with control or CTCF shRNA expression vectors (KD-1 and KD-2). (B) Real-time RT-PCR showing relative JUN and EGR1 mRNA levels in control or CTCF KD HeLa cells. (C) POL II binding across the IEGs, JUN and EGR1 loci, was mapped by POL II ChIP in control and CTCF KD HeLa cells at the paused state (minus EGF). POL II occupancy at the indicated genetic elements was measured using real-time PCR primers as denoted in the schematics showing EGR1 and JUN genes ( Table S1 ). Data shown are percent (%) of input at each genetic element. (D) Real-time RT-PCR showing relative EGR1 , JUN , NUP153 and CTCF mRNA levels were measured in NUP153 KD, CTCF KD and in CTCF/NUP153 KD HeLa cells. Values are mean ± standard deviation. Relative mRNA levels were normalized using GAPDH mRNA levels. Student’s t-test was applied to calculate significance. * p

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Quantitative RT-PCR, Transfection, shRNA, Expressing, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

    NUP153 is critical for CTCF and cohesin binding at the IEG cis -regulatory sites and across the IEG loci at the paused state. NUP153, CTCF and SMC3 occupancy across the JUN (left) and EGR1 (right) genetic elements were examined by ChIP real-time PCR at the paused state (minus EGF) and upon transcription induction (15 min EGF) in control and NUP153 KD HeLa cells. Position of PCR primers are denoted as numbers in the schematics showing EGR1 and JUN genes (See Table S1 for primer sequences). Data shown are percent (%) of input at each genetic element. Values are mean ± standard deviation. Student’s t-test was applied to calculate significance. * p

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: NUP153 is critical for CTCF and cohesin binding at the IEG cis -regulatory sites and across the IEG loci at the paused state. NUP153, CTCF and SMC3 occupancy across the JUN (left) and EGR1 (right) genetic elements were examined by ChIP real-time PCR at the paused state (minus EGF) and upon transcription induction (15 min EGF) in control and NUP153 KD HeLa cells. Position of PCR primers are denoted as numbers in the schematics showing EGR1 and JUN genes (See Table S1 for primer sequences). Data shown are percent (%) of input at each genetic element. Values are mean ± standard deviation. Student’s t-test was applied to calculate significance. * p

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

    NUP153 mediates CTCF and cohesin binding at cis -regulatory elements and TAD boundaries in mouse ES cells. (A) Distribution of NUP153 peaks in mouse ES cells. Peaks are categorized as promoters (×2kb from TSS to +100 bp from TSS), gene body (+100bp from TSS to +1kb from transcription termination site (TTS)), intergenic sites (

    Journal: bioRxiv

    Article Title: Nucleoporin 153 links nuclear pore complex to chromatin architecture by mediating CTCF and cohesin binding at cis-regulatory elements and TAD boundaries

    doi: 10.1101/2020.02.04.934398

    Figure Lengend Snippet: NUP153 mediates CTCF and cohesin binding at cis -regulatory elements and TAD boundaries in mouse ES cells. (A) Distribution of NUP153 peaks in mouse ES cells. Peaks are categorized as promoters (×2kb from TSS to +100 bp from TSS), gene body (+100bp from TSS to +1kb from transcription termination site (TTS)), intergenic sites (

    Article Snippet: FLAG-hNUP153 (human) or FLAG-mNUP153 (mouse) expression vectors were constructed by amplifying full length human NUP153 or mouse NUP153 cDNA using human NUP153 cDNA (Origene, SC116943) or mouse NUP143 cDNA (ATCC, IMAGE clone ID: 6516328) clones, respectively.

    Techniques: Binding Assay

    RIF1-L promotes resistance to Aphidicolin treatment but RIF1-S cannot (A) Illustration of RIF1 exon structure and construction of HCT116 mAC-RIF1-L and mAC-RIF1-S cell lines. Constructs containing cDNA encoding the C-terminal portion of either RIF1-L or RIF1-S were inserted at the end of exon 29, by CRISPR-mediated integration of donor plasmids into the HCT116 mAC-RIF1 cell line. (B) Expression of RIF1 in HCT116 mAC-RIF1, mAC-RIF1-L, mAC-RIF1-S and RIF1 KO cell lines. Whole cell extracts were harvested for western blotting with anti-RIF1 antibody. Loading control shows tubulin. (C) CFA comparing resistance to Aphidicolin of mAC-RIF1(black bars), mAC-RIF1-L (red bars), mAC-RIF1-S (grey bars), and RIF1-KO (open bars) cell lines. *p

    Journal: bioRxiv

    Article Title: RIF1-Long promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

    doi: 10.1101/859199

    Figure Lengend Snippet: RIF1-L promotes resistance to Aphidicolin treatment but RIF1-S cannot (A) Illustration of RIF1 exon structure and construction of HCT116 mAC-RIF1-L and mAC-RIF1-S cell lines. Constructs containing cDNA encoding the C-terminal portion of either RIF1-L or RIF1-S were inserted at the end of exon 29, by CRISPR-mediated integration of donor plasmids into the HCT116 mAC-RIF1 cell line. (B) Expression of RIF1 in HCT116 mAC-RIF1, mAC-RIF1-L, mAC-RIF1-S and RIF1 KO cell lines. Whole cell extracts were harvested for western blotting with anti-RIF1 antibody. Loading control shows tubulin. (C) CFA comparing resistance to Aphidicolin of mAC-RIF1(black bars), mAC-RIF1-L (red bars), mAC-RIF1-S (grey bars), and RIF1-KO (open bars) cell lines. *p

    Article Snippet: cDNA analysis of RIF1 splice variants A human cDNA panel covering 48 major tissues was obtained from Insight Biotechnology (Origene HMRT104).

    Techniques: Construct, CRISPR, Expressing, Western Blot