• Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    PARP Antibody
    Description:
    PARP a 116 kDa nuclear poly ADP ribose polymerase appears to be involved in DNA repair in response to environmental stress 1 This protein can be cleaved by many ICE like caspases in vitro 2 3 and is one of the main cleavage targets of caspase 3 in vivo 4 5 In human PARP the cleavage occurs between Asp214 and Gly215 which separates the PARP amino terminal DNA binding domain 24 kDa from the carboxy terminal catalytic domain 89 kDa 2 4 PARP helps cells to maintain their viability cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis 6
    Catalog Number:
    9542
    Price:
    None
    Applications:
    Western Blot
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the caspase cleavage site in PARP. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc p21
    Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors <t>(p21,</t> 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.
    PARP a 116 kDa nuclear poly ADP ribose polymerase appears to be involved in DNA repair in response to environmental stress 1 This protein can be cleaved by many ICE like caspases in vitro 2 3 and is one of the main cleavage targets of caspase 3 in vivo 4 5 In human PARP the cleavage occurs between Asp214 and Gly215 which separates the PARP amino terminal DNA binding domain 24 kDa from the carboxy terminal catalytic domain 89 kDa 2 4 PARP helps cells to maintain their viability cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis 6
    https://www.bioz.com/result/p21/product/Cell Signaling Technology Inc
    Average 99 stars, based on 544 article reviews
    Price from $9.99 to $1999.99
    p21 - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Epstein-Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells"

    Article Title: Epstein-Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017809

    Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors (p21, 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.
    Figure Legend Snippet: Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors (p21, 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Expressing

    2) Product Images from "Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia"

    Article Title: Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia

    Journal: Scientific Reports

    doi: 10.1038/srep26510

    NMHC induced cell cycle arrest and cell apoptotic death in AML. ( a ) Analysis of cell cycle distributions in NMHC-treated cells. Cells were treated with various doses of NMHC for 24 h, followed by cell cycle analysis. The percentages of G0-G1 phases are shown in each graph. Effect of NMHC on cell cycle-relevant proteins was shown in the bottom. Cyclin B1 and p21 were detected by immunoblotting after 24 h treatments. ( b ) Analysis of apoptotic cell death. Cells were treated with various doses of NMHC for 24 h, stained with Annexin V-FITC/PI and analyzed by flow cytometry. Apoptosis-relevant proteins PARP and cleaved Caspase-3 were assessed by immunoblotting after DMSO or NMHC (1 μM) treatments for 24 h. Above all, representative data from three independent experiments are shown. β-actin is detected as the internal reference.
    Figure Legend Snippet: NMHC induced cell cycle arrest and cell apoptotic death in AML. ( a ) Analysis of cell cycle distributions in NMHC-treated cells. Cells were treated with various doses of NMHC for 24 h, followed by cell cycle analysis. The percentages of G0-G1 phases are shown in each graph. Effect of NMHC on cell cycle-relevant proteins was shown in the bottom. Cyclin B1 and p21 were detected by immunoblotting after 24 h treatments. ( b ) Analysis of apoptotic cell death. Cells were treated with various doses of NMHC for 24 h, stained with Annexin V-FITC/PI and analyzed by flow cytometry. Apoptosis-relevant proteins PARP and cleaved Caspase-3 were assessed by immunoblotting after DMSO or NMHC (1 μM) treatments for 24 h. Above all, representative data from three independent experiments are shown. β-actin is detected as the internal reference.

    Techniques Used: Cell Cycle Assay, Staining, Flow Cytometry, Cytometry

    3) Product Images from "Autophagy controls mesenchymal stem cell properties and senescence during bone aging, et al. Autophagy controls mesenchymal stem cell properties and senescence during bone aging"

    Article Title: Autophagy controls mesenchymal stem cell properties and senescence during bone aging, et al. Autophagy controls mesenchymal stem cell properties and senescence during bone aging

    Journal: Aging Cell

    doi: 10.1111/acel.12709

    Aged bone marrow‐derived mesenchymal stem cells ( BMMSC s) showed degenerative properties in senile osteoporosis compared with young BMMSC s. (a) Micro‐ CT analysis of trabecular bone mass in the femora of 3‐month‐old (young) and 16‐month‐old (aged) mice. Quantitative analysis was performed including (b) bone mineral density ( BMD ) ( N = 3), (c) trabecular bone volume ( BV / TV ) ( N = 3), (d) trabecular number (Tb.N) ( N = 3), (e) trabecular thickness (Tb.Th) ( N = 3) and (f) trabecular space (Tb.Sp) ( N = 3). At the cellular level: (g, h) senescence‐associated β‐galactosidase analysis of cultured BMMSC s derived from young and aged mice and quantitative analysis of positive cells. Scale bar = 100 μm. (i, j) The protein level of senescence markers (p53, p21, p16) of young and aged cells were determined by Western blot. (k, l) Alizarin Red staining of young and aged BMMSC s. OS = osteogenically induced. (m, n) Oil Red staining of young BMMSC s and aged BMMSC s. AD = adipogenically induced. Scale bar = 100 μm. Results are presented as means ± SD . n = 3. * p
    Figure Legend Snippet: Aged bone marrow‐derived mesenchymal stem cells ( BMMSC s) showed degenerative properties in senile osteoporosis compared with young BMMSC s. (a) Micro‐ CT analysis of trabecular bone mass in the femora of 3‐month‐old (young) and 16‐month‐old (aged) mice. Quantitative analysis was performed including (b) bone mineral density ( BMD ) ( N = 3), (c) trabecular bone volume ( BV / TV ) ( N = 3), (d) trabecular number (Tb.N) ( N = 3), (e) trabecular thickness (Tb.Th) ( N = 3) and (f) trabecular space (Tb.Sp) ( N = 3). At the cellular level: (g, h) senescence‐associated β‐galactosidase analysis of cultured BMMSC s derived from young and aged mice and quantitative analysis of positive cells. Scale bar = 100 μm. (i, j) The protein level of senescence markers (p53, p21, p16) of young and aged cells were determined by Western blot. (k, l) Alizarin Red staining of young and aged BMMSC s. OS = osteogenically induced. (m, n) Oil Red staining of young BMMSC s and aged BMMSC s. AD = adipogenically induced. Scale bar = 100 μm. Results are presented as means ± SD . n = 3. * p

    Techniques Used: Derivative Assay, Micro-CT, Mouse Assay, Cell Culture, Western Blot, Staining

    4) Product Images from "Sensitivity to cdk1-inhibition is modulated by p53 status in preclinical models of embryonal tumors"

    Article Title: Sensitivity to cdk1-inhibition is modulated by p53 status in preclinical models of embryonal tumors

    Journal: Oncotarget

    doi:

    Inhibition of cdk1 by a small molecule inhibitor, RO-3306, induces apoptosis and causes an activation of the p53 signaling pathway A. Inhibition of cdk1 by RO-3306 caused decreased cell viability after 48 h in all NB cell lines investigated and also in the rhabdomyosarcoma cell line RH-41 in a concentration dependent manner. The human fibroblast cell line, NHDF, and the cell lines harboring p53 mutation (SK-N-FI, RH-41) were more resistant to RO-3306 than the tumor cell lines with p53 wild type. B. Cell cycle distribution was analyzed 24 h and 48 h after RO-3306 treatment. Only cells harboring a p53 mutation (SK-N-FI, NLF) presented with significantly higher fraction of G2 phase (left side), while all cell lines had significantly elevated subG1 indicative of apoptosis. C. Significant induction of apoptosis by RO-3306 could be confirmed in all cell lines by a colorimetric assay detecting nucleosomes. D. Activation of the p53 pathway in p53 wt cells shown by increased p53 and p21 protein levels. Significant differences between untreated cells and inhibitor treated cells are depicted in the entire diagram using the following code “***”: p
    Figure Legend Snippet: Inhibition of cdk1 by a small molecule inhibitor, RO-3306, induces apoptosis and causes an activation of the p53 signaling pathway A. Inhibition of cdk1 by RO-3306 caused decreased cell viability after 48 h in all NB cell lines investigated and also in the rhabdomyosarcoma cell line RH-41 in a concentration dependent manner. The human fibroblast cell line, NHDF, and the cell lines harboring p53 mutation (SK-N-FI, RH-41) were more resistant to RO-3306 than the tumor cell lines with p53 wild type. B. Cell cycle distribution was analyzed 24 h and 48 h after RO-3306 treatment. Only cells harboring a p53 mutation (SK-N-FI, NLF) presented with significantly higher fraction of G2 phase (left side), while all cell lines had significantly elevated subG1 indicative of apoptosis. C. Significant induction of apoptosis by RO-3306 could be confirmed in all cell lines by a colorimetric assay detecting nucleosomes. D. Activation of the p53 pathway in p53 wt cells shown by increased p53 and p21 protein levels. Significant differences between untreated cells and inhibitor treated cells are depicted in the entire diagram using the following code “***”: p

    Techniques Used: Inhibition, Activation Assay, Concentration Assay, Mutagenesis, Colorimetric Assay

    5) Product Images from "MTA1 promotes proliferation and invasion in human gastric cancer cells"

    Article Title: MTA1 promotes proliferation and invasion in human gastric cancer cells

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S85383

    MTA1-induced upregulation of cyclin D1 and downregulation of p21. Notes: SGC7901 ( A ) and BGC823 ( B ) cells were transfected with control (Ctrl) vector and MTA1 expression vector. After 48 hours of transfection, the protein levels of cyclin D1 and p21 were measured by Western blot analysis. MTA1 expression vectors were transiently transfected into SGC7901 ( C ) and BGC823 ( D ) cells, and the mRNA level was estimated by real-time polymerase chain reaction analysis. GAPDH was used as a loading Ctrl. Bars represent the mean ± standard deviation of three independent experiments. * P
    Figure Legend Snippet: MTA1-induced upregulation of cyclin D1 and downregulation of p21. Notes: SGC7901 ( A ) and BGC823 ( B ) cells were transfected with control (Ctrl) vector and MTA1 expression vector. After 48 hours of transfection, the protein levels of cyclin D1 and p21 were measured by Western blot analysis. MTA1 expression vectors were transiently transfected into SGC7901 ( C ) and BGC823 ( D ) cells, and the mRNA level was estimated by real-time polymerase chain reaction analysis. GAPDH was used as a loading Ctrl. Bars represent the mean ± standard deviation of three independent experiments. * P

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    6) Product Images from "Metformin Suppresses the Proliferation and Promotes the Apoptosis of Colon Cancer Cells Through Inhibiting the Expression of Long Noncoding RNA-UCA1"

    Article Title: Metformin Suppresses the Proliferation and Promotes the Apoptosis of Colon Cancer Cells Through Inhibiting the Expression of Long Noncoding RNA-UCA1

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S245091

    LncRNA-UCA1 knockdown facilitates the anticancer effects of metformin against colon cancer cells in vitro. ( A ) qRT-PCR was conducted to evaluate the effects of MET or UCA1 knockdown or MET + UCA1 knockdown on the expression of lncRNA-UCA1 in the SW480 and SW620 cells in vitro. ( B ) CCK-8 cellular viability assays were used to test the antiproliferative effects of MET or UCA1 knockdown or MET + UCA1 knockdown on colon cancer cells in vitro. ( C ) A flow cytometry-based method was conducted to test the effects of MET or UCA1 knockdown or MET + UCA1 knockdown on the cellular apoptosis of the SW480 and SW620 cells in vitro. ( D ) Western blotting was conducted to evaluate the effects of MET or UCA1 knockdown or MET + UCA1 knockdown on the expression of two proliferation-related markers (p21 and PCNA) and three apoptosis-related markers (Bax, Bcl-2 and cleaved caspase3) in colon cancer cells. *p-value
    Figure Legend Snippet: LncRNA-UCA1 knockdown facilitates the anticancer effects of metformin against colon cancer cells in vitro. ( A ) qRT-PCR was conducted to evaluate the effects of MET or UCA1 knockdown or MET + UCA1 knockdown on the expression of lncRNA-UCA1 in the SW480 and SW620 cells in vitro. ( B ) CCK-8 cellular viability assays were used to test the antiproliferative effects of MET or UCA1 knockdown or MET + UCA1 knockdown on colon cancer cells in vitro. ( C ) A flow cytometry-based method was conducted to test the effects of MET or UCA1 knockdown or MET + UCA1 knockdown on the cellular apoptosis of the SW480 and SW620 cells in vitro. ( D ) Western blotting was conducted to evaluate the effects of MET or UCA1 knockdown or MET + UCA1 knockdown on the expression of two proliferation-related markers (p21 and PCNA) and three apoptosis-related markers (Bax, Bcl-2 and cleaved caspase3) in colon cancer cells. *p-value

    Techniques Used: In Vitro, Quantitative RT-PCR, Expressing, CCK-8 Assay, Flow Cytometry, Western Blot

    7) Product Images from "The human positive cofactor 4 promotes androgen-independent prostate cancer development and progression through HIF-1α/β-catenin pathway"

    Article Title: The human positive cofactor 4 promotes androgen-independent prostate cancer development and progression through HIF-1α/β-catenin pathway

    Journal: American Journal of Cancer Research

    doi:

    PC4 exerts the oncogenic functions through enhancing β-catenin signaling. (A) Immunofluorescent staining for β-catenin expression in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls. Scale bar represents 50 µm. (B) Western Blot assay for β-catenin expression in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls. (C) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell cycle distribution was determined by flow cytometry. (D) Statistical analysis of the data derived from (C). Experiments were repeated three times independently. (E) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell migration capacity was determined by scratch-wound assay. Scale bar represents 200 µm. (F) Statistical analysis of the data derived from (E). Experiments were repeated three times independently. (G) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell viability was determined by CCK-8 assay at 24h, 48h, 72h and 96h. (H) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell invasive capacity was determined by transwell assay. Scale bar represents 100 µm. (I) Statistical analysis of the data derived from (H). Experiments were repeated three times independently. (J) After overexpression of β-catenin in PC4 knockdown PC3 cells, the protein levels of c-Myc, P21, Cyclin D, Cyclin E, Snail and E-cadherin were determined by Western Blot. All data indicate the mean ± SD. *P
    Figure Legend Snippet: PC4 exerts the oncogenic functions through enhancing β-catenin signaling. (A) Immunofluorescent staining for β-catenin expression in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls. Scale bar represents 50 µm. (B) Western Blot assay for β-catenin expression in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls. (C) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell cycle distribution was determined by flow cytometry. (D) Statistical analysis of the data derived from (C). Experiments were repeated three times independently. (E) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell migration capacity was determined by scratch-wound assay. Scale bar represents 200 µm. (F) Statistical analysis of the data derived from (E). Experiments were repeated three times independently. (G) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell viability was determined by CCK-8 assay at 24h, 48h, 72h and 96h. (H) After overexpression of β-catenin in PC4 knockdown PC3 cells, cell invasive capacity was determined by transwell assay. Scale bar represents 100 µm. (I) Statistical analysis of the data derived from (H). Experiments were repeated three times independently. (J) After overexpression of β-catenin in PC4 knockdown PC3 cells, the protein levels of c-Myc, P21, Cyclin D, Cyclin E, Snail and E-cadherin were determined by Western Blot. All data indicate the mean ± SD. *P

    Techniques Used: Staining, Expressing, Western Blot, Over Expression, Flow Cytometry, Cytometry, Derivative Assay, Migration, Scratch Wound Assay Assay, CCK-8 Assay, Transwell Assay

    Schematic illustration for the potential mechanisms of PC4 in AIPC progression. In AIPC, PC4 can promote the expression of HIF-1α and enhance β-catenin signaling. On one hand, β-catenin activates c-Myc and modulates p21 mediated cyclin-dependent kinase-pRb pathway, that accelerates G1/S phase transition to promote cell proliferation in AIPC. On other hand, β-catenin regulates the EMT-related gene and promotes metastasis in AIPC.
    Figure Legend Snippet: Schematic illustration for the potential mechanisms of PC4 in AIPC progression. In AIPC, PC4 can promote the expression of HIF-1α and enhance β-catenin signaling. On one hand, β-catenin activates c-Myc and modulates p21 mediated cyclin-dependent kinase-pRb pathway, that accelerates G1/S phase transition to promote cell proliferation in AIPC. On other hand, β-catenin regulates the EMT-related gene and promotes metastasis in AIPC.

    Techniques Used: Expressing, Sublimation

    Loss of PC4 induces cell cycle arrest at the G1-to-S phase transition through inhibiting c-Myc/P21 pathway. (A) Cell cycle distribution in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls was determined by flow cytometry. (B) Statistical analysis of the data derived from (A). Experiments were repeated three times independently. (C) Apoptosis rate in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls was determined by flow cytometry. (D) Statistical analysis of the data derived from (C). Experiments were repeated three times independently. (E) The protein levels of c-Myc, P21, Cyclin D, Cyclin E, CDK6, Rb and phosphorylated Rb (ser807/811) were determined by Western Blot in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls. All data indicate the mean ± SD. *P
    Figure Legend Snippet: Loss of PC4 induces cell cycle arrest at the G1-to-S phase transition through inhibiting c-Myc/P21 pathway. (A) Cell cycle distribution in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls was determined by flow cytometry. (B) Statistical analysis of the data derived from (A). Experiments were repeated three times independently. (C) Apoptosis rate in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls was determined by flow cytometry. (D) Statistical analysis of the data derived from (C). Experiments were repeated three times independently. (E) The protein levels of c-Myc, P21, Cyclin D, Cyclin E, CDK6, Rb and phosphorylated Rb (ser807/811) were determined by Western Blot in stable PC4 knockdown cell lines (PC3 and DU145 cells) and controls. All data indicate the mean ± SD. *P

    Techniques Used: Sublimation, Flow Cytometry, Cytometry, Derivative Assay, Western Blot

    8) Product Images from "HBP1-mediated Regulation of p21 Protein through the Mdm2/p53 and TCF4/EZH2 Pathways and Its Impact on Cell Senescence and Tumorigenesis *"

    Article Title: HBP1-mediated Regulation of p21 Protein through the Mdm2/p53 and TCF4/EZH2 Pathways and Its Impact on Cell Senescence and Tumorigenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.714147

    Transcription factor HBP1 promotes p21 transcription, probably by regulating the expression of p53 and EZH2. A , HBP1 and p21 are down-regulated at the mRNA level in lung adenocarcinoma according to bioinformatics analysis. In the rank-based gene expression curves, the x axis represents expressional intensity reflected by rank scores, whereas the y axis indicates sample percentiles at each rank score. B , HBP1 overexpression increases p53 and p21 expression and decreases EZH2 expression. The protein levels of HBP1, p53, p21, Mdm2, and EZH2 were measured by Western blotting in A549, HepG2, or U2OS cells stably transfected with PITA or PITA-HA-HBP1 through lentiviral infection. GAPDH was used as a loading control ( left panel ). The mRNA levels of p53, p21, Mdm2, and EZH2 were measured by real-time PCR in A549 cells stably transfected with PITA or PITA-HA-HBP1 through lentiviral infection. Results are representative of three independent experiments, and values are the mean ± S.E. *, p
    Figure Legend Snippet: Transcription factor HBP1 promotes p21 transcription, probably by regulating the expression of p53 and EZH2. A , HBP1 and p21 are down-regulated at the mRNA level in lung adenocarcinoma according to bioinformatics analysis. In the rank-based gene expression curves, the x axis represents expressional intensity reflected by rank scores, whereas the y axis indicates sample percentiles at each rank score. B , HBP1 overexpression increases p53 and p21 expression and decreases EZH2 expression. The protein levels of HBP1, p53, p21, Mdm2, and EZH2 were measured by Western blotting in A549, HepG2, or U2OS cells stably transfected with PITA or PITA-HA-HBP1 through lentiviral infection. GAPDH was used as a loading control ( left panel ). The mRNA levels of p53, p21, Mdm2, and EZH2 were measured by real-time PCR in A549 cells stably transfected with PITA or PITA-HA-HBP1 through lentiviral infection. Results are representative of three independent experiments, and values are the mean ± S.E. *, p

    Techniques Used: Expressing, Over Expression, Western Blot, Stable Transfection, Transfection, Infection, Real-time Polymerase Chain Reaction

    Model for the regulation of p21 by HBP1 and its role in senescence induction and tumor inhibition. HBP1 can positively regulate p21 expression through both the p53/Mdm2 pathway and TCF4/EZH2 pathway. HBP1 binds Mdm2 through the repression domain and inhibits the interaction between p53 and Mdm2, thus abrogating Mdm2-mediated p53 degradation. The stabilized p53 then activates p21 transcription and leads to p21 elevation. HBP1 can also bind to TCF4 and inhibit the activation of EZH2 transcription by TCF4, resulting in a decrease in the level of H3K27me3 on the p21 promoter. The H3K27me3 hypomethylation state of the p21 promoter enhances p53 binding and p21 transcriptional activation. Overall, HBP1-mediated activation of p21 through Mdm2/p53 and TCF4/EZH2 pathways synergistically contributes to HBP1-induced premature senescence and tumor inhibition.
    Figure Legend Snippet: Model for the regulation of p21 by HBP1 and its role in senescence induction and tumor inhibition. HBP1 can positively regulate p21 expression through both the p53/Mdm2 pathway and TCF4/EZH2 pathway. HBP1 binds Mdm2 through the repression domain and inhibits the interaction between p53 and Mdm2, thus abrogating Mdm2-mediated p53 degradation. The stabilized p53 then activates p21 transcription and leads to p21 elevation. HBP1 can also bind to TCF4 and inhibit the activation of EZH2 transcription by TCF4, resulting in a decrease in the level of H3K27me3 on the p21 promoter. The H3K27me3 hypomethylation state of the p21 promoter enhances p53 binding and p21 transcriptional activation. Overall, HBP1-mediated activation of p21 through Mdm2/p53 and TCF4/EZH2 pathways synergistically contributes to HBP1-induced premature senescence and tumor inhibition.

    Techniques Used: Inhibition, Expressing, Activation Assay, Binding Assay

    HBP1 also enhances p21 transcription in the absence of p53. A–C , HBP1 regulates p21 and EZH2 expression in the absence of p53. A , H1299 cells were transfected with pcDNA3 or pcDNA3-HA-HBP1. The protein levels of p21, Mdm2, and EZH2 were detected by Western blotting ( left panel ), and mRNA from the same cells was extracted and analyzed by real-time PCR. Results are representative of three independent experiments, and values are the mean ± S.E. *, p
    Figure Legend Snippet: HBP1 also enhances p21 transcription in the absence of p53. A–C , HBP1 regulates p21 and EZH2 expression in the absence of p53. A , H1299 cells were transfected with pcDNA3 or pcDNA3-HA-HBP1. The protein levels of p21, Mdm2, and EZH2 were detected by Western blotting ( left panel ), and mRNA from the same cells was extracted and analyzed by real-time PCR. Results are representative of three independent experiments, and values are the mean ± S.E. *, p

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

    HBP1 promotes p21 transcription by enhancing p53 protein stability. A , HBP1 prolongs the half-life of p53. A549 cells were stably transfected with PITA, PITA-HA-HBP1 or pLL3.7, and pLL3.7-shHBP1 through lentiviral infection. Cells were incubated with the protein translation inhibitor cycloheximide ( CHX ) for 0, 20, 40, 60, or 80 min before harvest. P53 and Mdm2 protein levels were detected by Western blotting. GAPDH was used as the loading control for this turnover experiment ( top panel ). Densitometry is plotted for the average ± S.D. of three independent experiments ( bottom panel ). B , HBP1 does not elevate p53 levels in the presence of MG132. H1299 cells were cotransfected with p53 and GFP with or without HBP1. 42 h after transfection, cells were incubated with ( third and fourth lanes ) or without ( first and second lanes ) MG132 for another 6 h. p53 protein was detected by Western blotting. Level of GFP is shown as equal transfection efficiency. C , overexpression of HBP1 enhances the binding of p53 to the p21 promoter. H1299 cells were transfected with HA-tagged p53 and pcDNA3, pcDNA3-HA-HBP1, pcDNA3.1-His-EZH2, or both pcDNA3-HA-HBP1 and pcDNA3.1-His-EZH2. 24 h after transfection, cells were lysed for ChIP assay. The lysates were incubated with anti-p53 antibody or control IgG. The precipitated DNA fragments were amplified with specific oligonucleotides for the p21 promoter by real-time PCR. Results are representative of three independent experiments, and values are the mean ± S.E. *, p
    Figure Legend Snippet: HBP1 promotes p21 transcription by enhancing p53 protein stability. A , HBP1 prolongs the half-life of p53. A549 cells were stably transfected with PITA, PITA-HA-HBP1 or pLL3.7, and pLL3.7-shHBP1 through lentiviral infection. Cells were incubated with the protein translation inhibitor cycloheximide ( CHX ) for 0, 20, 40, 60, or 80 min before harvest. P53 and Mdm2 protein levels were detected by Western blotting. GAPDH was used as the loading control for this turnover experiment ( top panel ). Densitometry is plotted for the average ± S.D. of three independent experiments ( bottom panel ). B , HBP1 does not elevate p53 levels in the presence of MG132. H1299 cells were cotransfected with p53 and GFP with or without HBP1. 42 h after transfection, cells were incubated with ( third and fourth lanes ) or without ( first and second lanes ) MG132 for another 6 h. p53 protein was detected by Western blotting. Level of GFP is shown as equal transfection efficiency. C , overexpression of HBP1 enhances the binding of p53 to the p21 promoter. H1299 cells were transfected with HA-tagged p53 and pcDNA3, pcDNA3-HA-HBP1, pcDNA3.1-His-EZH2, or both pcDNA3-HA-HBP1 and pcDNA3.1-His-EZH2. 24 h after transfection, cells were lysed for ChIP assay. The lysates were incubated with anti-p53 antibody or control IgG. The precipitated DNA fragments were amplified with specific oligonucleotides for the p21 promoter by real-time PCR. Results are representative of three independent experiments, and values are the mean ± S.E. *, p

    Techniques Used: Stable Transfection, Transfection, Infection, Incubation, Western Blot, Over Expression, Binding Assay, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    HBP1-mediated activation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to HBP1-induced premature senescence and tumor inhibition. A , the protein levels of HBP1, p53, EZH2, and p21 during replicative senescence. Shown is an immunoblot ( IB ) of WI-38 cells in young and old stages. The expression of endogenous HBP1, p53, β-catenin, EZH2, Wnt2, p21, and p16 is shown. GAPDH was used as a loading control ( left panel ). WI-38 cells were lysed with IP lysis buffer and then subjected to immunoprecipitation with anti-HBP1 antibody followed by Western blotting analysis with anti-Mdm2 and anti-TCF4 antibodies ( right panel ). B , exogenous EZH2 inhibits p21 expression. The protein levels of EZH2, p53, and p21 were measured by immunoblotting in WI-38 cells (PD20) retrovirally transduced with EZH2 or control vector. C , EZH2 attenuates HBP1 elevation of p21 when p53 is knocked down by shRNA. The levels of EZH2, p53, p21, and GAPDH (as a control) were determined by Western blotting of WI-38 cells retrovirally transduced with control vector, HBP1, HBP1 + p53shRNA, or HBP1 + EZH2 + p53shRNA. D and E , EZH2 attenuates HBP1-induced cell growth arrest when p53 is knocked down by shRNA. MTT ( D ) and BrdU ( E ) incorporation assays were conducted with WI-38 cells retrovirally transduced with control vector, HBP1, HBP1 + p53shRNA, or HBP1 + EZH2 + p53shRNA. The means ± S.E. for three independent experiments are shown. OD , optical density. *, p
    Figure Legend Snippet: HBP1-mediated activation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to HBP1-induced premature senescence and tumor inhibition. A , the protein levels of HBP1, p53, EZH2, and p21 during replicative senescence. Shown is an immunoblot ( IB ) of WI-38 cells in young and old stages. The expression of endogenous HBP1, p53, β-catenin, EZH2, Wnt2, p21, and p16 is shown. GAPDH was used as a loading control ( left panel ). WI-38 cells were lysed with IP lysis buffer and then subjected to immunoprecipitation with anti-HBP1 antibody followed by Western blotting analysis with anti-Mdm2 and anti-TCF4 antibodies ( right panel ). B , exogenous EZH2 inhibits p21 expression. The protein levels of EZH2, p53, and p21 were measured by immunoblotting in WI-38 cells (PD20) retrovirally transduced with EZH2 or control vector. C , EZH2 attenuates HBP1 elevation of p21 when p53 is knocked down by shRNA. The levels of EZH2, p53, p21, and GAPDH (as a control) were determined by Western blotting of WI-38 cells retrovirally transduced with control vector, HBP1, HBP1 + p53shRNA, or HBP1 + EZH2 + p53shRNA. D and E , EZH2 attenuates HBP1-induced cell growth arrest when p53 is knocked down by shRNA. MTT ( D ) and BrdU ( E ) incorporation assays were conducted with WI-38 cells retrovirally transduced with control vector, HBP1, HBP1 + p53shRNA, or HBP1 + EZH2 + p53shRNA. The means ± S.E. for three independent experiments are shown. OD , optical density. *, p

    Techniques Used: Activation Assay, Inhibition, Expressing, Lysis, Immunoprecipitation, Western Blot, Transduction, Plasmid Preparation, shRNA, MTT Assay

    9) Product Images from "Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer"

    Article Title: Long noncoding AGAP2-AS1 is activated by SP1 and promotes cell proliferation and invasion in gastric cancer

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0420-4

    AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P
    Figure Legend Snippet: AGAP2-AS1 epigenetically suppresses P21 and E-cadherin by interacting with EZH2 and LSD1. a , b qRT-PCR analysis of the expression levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with EZH2, LSD1, or negative control siRNAs. c , d ChIP-qPCR analysis of EZH2, H3K27me3, LSD1, and H3K4me2 occupancy in the P21 and E-cadherin promoters in the BGC823 and AGS cells after transfection with AGAP2-AS1 or NC siRNA. IgG was used as a negative control. e The relationship between AGAP2-AS1 expression and P21/E-cadherin in the GC tissues was analyzed. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P
    Figure Legend Snippet: AGAP2-AS1 interacting with EZH2 and LSD1 in the GC cells. a The distribution of AGAP2-AS1 levels in the cytoplasmic or nuclear fraction of GC cell lines was determined by qRT-PCR. U1 was used as a nuclear control; GAPDH was used as a cytoplasmic control. b The AGAP2-AS1 RNA levels in EZH2, LSD1, SUZ12, CoREST, and HuR immunoprecipitates were determined by qRT-PCR, and data are presented as fold enrichment relative to IgG immunoprecipitates. c EZH2 and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 RNA were determined by Western blot. HuR protein immunoprecipitates with AR RNA were used as a positive control. d qRT-PCR analysis of the expression levels of KLF2, LATS1, and LATS2, and others in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. e Western blot analysis of the protein levels of P21 and E-cadherin in the BGC823 and AGS cells after transfection with AGAP2-AS1 or negative control siRNAs. f Immunofluorescence staining analysis of E-cadherin in BGC823 cells after transfection with AGAP2-AS1 or negative control siRNAs. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Positive Control, Expressing, Transfection, Negative Control, Immunofluorescence, Staining

    10) Product Images from "CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer"

    Article Title: CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24231

    Negative effects of CXCR2 on romidepsin-induced p21 protein expression via Akt-Mdm2 axis in a p53-independent manner ( A ) Dose-dependent effects of romidepsin on Akt, Mdm2 and p21 protein expression in p53 null SKOV-3 cells. Cells was treated with 0, 4, 8, 16, 32 and 64 nM romidepsin for 24 h. ( B ) Effects of silencing Akt1 and MDM2 on romidepsin-induced p21 protein expression in SKCXCR2 cells. ( C ) Effects of overexpressed Akt1 on romidepsin-induced p21 protein expression in SKOV-3 cells. β-actin was detected as an internal loading control of cell lysates. Cells was treated with 64 nM romidepsin for 24 h. ( D ) Schematic representation of molecular mechanism of CXCR2-mediated Akt-Mdm2 axis on cell cycle inhibitor p21 regulation in p53-dependent and independent manner in ovarian cancer cells. A representative result is shown from duplicated experiments.
    Figure Legend Snippet: Negative effects of CXCR2 on romidepsin-induced p21 protein expression via Akt-Mdm2 axis in a p53-independent manner ( A ) Dose-dependent effects of romidepsin on Akt, Mdm2 and p21 protein expression in p53 null SKOV-3 cells. Cells was treated with 0, 4, 8, 16, 32 and 64 nM romidepsin for 24 h. ( B ) Effects of silencing Akt1 and MDM2 on romidepsin-induced p21 protein expression in SKCXCR2 cells. ( C ) Effects of overexpressed Akt1 on romidepsin-induced p21 protein expression in SKOV-3 cells. β-actin was detected as an internal loading control of cell lysates. Cells was treated with 64 nM romidepsin for 24 h. ( D ) Schematic representation of molecular mechanism of CXCR2-mediated Akt-Mdm2 axis on cell cycle inhibitor p21 regulation in p53-dependent and independent manner in ovarian cancer cells. A representative result is shown from duplicated experiments.

    Techniques Used: Expressing

    Resistant effects of CXCR2 on the anti-tumor potential of romidepsin in p53-null cells ( A ) Inhibitory effects of CXCR2 on romidepsin-induced p21 protein in SKA and SKCXCR2cells. Cells were treated with romidepsin (0, 32 and 64 nM) for 24 h. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. ( B ) IC50 of romidepsin in SKA and SKCXCR2 cells. Cells were treated with 10–100 nM romidepsin for 48 h and then cell proliferation assay was performed. All data are shown as mean ± SE from triplicated experiments. * indicates a significant increase ( p ≤ 0.05) by Student’s t -test. ( C ) Clonogenic survival assay in SKA and SKCXCR2 after treatment of romidepsin (0, 32 and 64 nM) for 48 h. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. All data are shown as mean ± SE from triplicated experiments. Each SE is located within circles.
    Figure Legend Snippet: Resistant effects of CXCR2 on the anti-tumor potential of romidepsin in p53-null cells ( A ) Inhibitory effects of CXCR2 on romidepsin-induced p21 protein in SKA and SKCXCR2cells. Cells were treated with romidepsin (0, 32 and 64 nM) for 24 h. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. ( B ) IC50 of romidepsin in SKA and SKCXCR2 cells. Cells were treated with 10–100 nM romidepsin for 48 h and then cell proliferation assay was performed. All data are shown as mean ± SE from triplicated experiments. * indicates a significant increase ( p ≤ 0.05) by Student’s t -test. ( C ) Clonogenic survival assay in SKA and SKCXCR2 after treatment of romidepsin (0, 32 and 64 nM) for 48 h. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. All data are shown as mean ± SE from triplicated experiments. Each SE is located within circles.

    Techniques Used: Proliferation Assay, Clonogenic Cell Survival Assay

    The p53-dependent effects of nutlin-3 on cell proliferation and p21 levels in p53WT, mutant and null cells ( A ) The p53-dependent effect of nutlin-3 on cell proliferation in CXCR2-negative vs. positive cells with different p53 status. All data are shown as mean ± SE from triplicated experiments. # and * indicate a significant decrease and increase ( p ≤ 0.05) by ANOVA and Student’s t -test, respectively. ( B ) Time-dependent effect of nutlin-3 on p53 and its downstream p21 expression in p53WT A2780 cells. ( C ) Comparative induction of nutlin-3 on p53 and p21 protein levels in p53WT cells (AA/ACXCR2), p53-mutant (OVA/OVCXCR2) and p53-null (SKA/SKCXCR2) cells. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments.
    Figure Legend Snippet: The p53-dependent effects of nutlin-3 on cell proliferation and p21 levels in p53WT, mutant and null cells ( A ) The p53-dependent effect of nutlin-3 on cell proliferation in CXCR2-negative vs. positive cells with different p53 status. All data are shown as mean ± SE from triplicated experiments. # and * indicate a significant decrease and increase ( p ≤ 0.05) by ANOVA and Student’s t -test, respectively. ( B ) Time-dependent effect of nutlin-3 on p53 and its downstream p21 expression in p53WT A2780 cells. ( C ) Comparative induction of nutlin-3 on p53 and p21 protein levels in p53WT cells (AA/ACXCR2), p53-mutant (OVA/OVCXCR2) and p53-null (SKA/SKCXCR2) cells. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments.

    Techniques Used: Mutagenesis, Expressing

    Negative effects of CXCR2 on p21 promoter activity in a p53-dependent manner ( A ) Differential effects of CXCR2 on p21 promoter activity in p53WT A2780 cells, p53-mutant OVCAR-3 and p53-null SKOV-3 cells. ( B ) The p53-dependent inhibition of CXCR2 on p21 promoter activity in deleted constructs of p21 promoter in p53WT A2780 cells. All data are shown as mean ± SE from triplicated experiments. ( C ) Induced effects of CXCR2 on Mdm2 protein in p53-dependent p21 regulation. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. # indicates a significant increase ( p ≤ 0.05) in each pair by Student’s t -test.
    Figure Legend Snippet: Negative effects of CXCR2 on p21 promoter activity in a p53-dependent manner ( A ) Differential effects of CXCR2 on p21 promoter activity in p53WT A2780 cells, p53-mutant OVCAR-3 and p53-null SKOV-3 cells. ( B ) The p53-dependent inhibition of CXCR2 on p21 promoter activity in deleted constructs of p21 promoter in p53WT A2780 cells. All data are shown as mean ± SE from triplicated experiments. ( C ) Induced effects of CXCR2 on Mdm2 protein in p53-dependent p21 regulation. β-actin was detected as an internal loading control of cell lysates. A representative result is shown from duplicated experiments. # indicates a significant increase ( p ≤ 0.05) in each pair by Student’s t -test.

    Techniques Used: Activity Assay, Mutagenesis, Inhibition, Construct

    Inhibitory effects of CXCR2-derived Akt-Mdm2 axis on p53-dependent p21 protein expression in p53WT cells ( A ) Knockdown of Mdm2 increased p21 promoter activity and recovered the CXCR2-mediated downregulation of p21. ( B ) Confirmation of the knockdown of Mdm2 protein through western blot and the effect of knockdown of Mdm2 protein on p53-dependent p21 protein expression. ( C ) Comparison of Akt activation in AA and ACXCR2 cells by confocal imaging analysis. ( D ) Knockdown of Akt1 increased p21 luciferase activity and recovered the CXCR2-mediated downregulation of p21. ( E ) Confirmation of the knockdown of Akt1 through western blot and the effect of knockdown of Akt1 on Mdm2, p53 and p21 protein expression levels. ( F ) Overexpression of Akt1 decreased the p21 luciferase activity and further abated the CXCR2-mediated downregulation of p21. ( G ) Confirmation of the overexpression of Akt1 through western blotting and the effect of silencing Akt1 on Mdm2, p53 and p21 protein expression. β-actin was detected as an internal loading control of cell lysates. All data are shown as mean ± SE from triplicated experiments (A, D, F) and a representative result is shown from duplicated experiments (B, C, E, G). * and # indicate a significant increase and decrease ( p ≤ 0.05), respectively, by Student’s t -test.
    Figure Legend Snippet: Inhibitory effects of CXCR2-derived Akt-Mdm2 axis on p53-dependent p21 protein expression in p53WT cells ( A ) Knockdown of Mdm2 increased p21 promoter activity and recovered the CXCR2-mediated downregulation of p21. ( B ) Confirmation of the knockdown of Mdm2 protein through western blot and the effect of knockdown of Mdm2 protein on p53-dependent p21 protein expression. ( C ) Comparison of Akt activation in AA and ACXCR2 cells by confocal imaging analysis. ( D ) Knockdown of Akt1 increased p21 luciferase activity and recovered the CXCR2-mediated downregulation of p21. ( E ) Confirmation of the knockdown of Akt1 through western blot and the effect of knockdown of Akt1 on Mdm2, p53 and p21 protein expression levels. ( F ) Overexpression of Akt1 decreased the p21 luciferase activity and further abated the CXCR2-mediated downregulation of p21. ( G ) Confirmation of the overexpression of Akt1 through western blotting and the effect of silencing Akt1 on Mdm2, p53 and p21 protein expression. β-actin was detected as an internal loading control of cell lysates. All data are shown as mean ± SE from triplicated experiments (A, D, F) and a representative result is shown from duplicated experiments (B, C, E, G). * and # indicate a significant increase and decrease ( p ≤ 0.05), respectively, by Student’s t -test.

    Techniques Used: Derivative Assay, Expressing, Activity Assay, Western Blot, Activation Assay, Imaging, Luciferase, Over Expression

    Effects of romidepsin on p21 promoter activity in p53-null SKOV-3 cells ( A ) Effects of CXCR2 on romidepsin-induced p21 promoter activity in SKOV-3 cells. ( B ) Dose-dependent effects of romidepsin on p21 luciferase activity in SKOV-3 cells. Experiments were performed in triplicate and all data are shown as mean ± S.E. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. ( C ) Effects of romidepsin on p21 promoter activity in deleted constructs of p21 promoter p53 response element in p53-null SKOV-3 cells. All data are shown as mean ± SE from triplicated experiments. * indicates a statistical significance ( p ≤ 0.05) by Student’s t -test.
    Figure Legend Snippet: Effects of romidepsin on p21 promoter activity in p53-null SKOV-3 cells ( A ) Effects of CXCR2 on romidepsin-induced p21 promoter activity in SKOV-3 cells. ( B ) Dose-dependent effects of romidepsin on p21 luciferase activity in SKOV-3 cells. Experiments were performed in triplicate and all data are shown as mean ± S.E. * ( p ≤ 0.05) in each group by ANOVA and Tukey’s pairwise comparisons. ( C ) Effects of romidepsin on p21 promoter activity in deleted constructs of p21 promoter p53 response element in p53-null SKOV-3 cells. All data are shown as mean ± SE from triplicated experiments. * indicates a statistical significance ( p ≤ 0.05) by Student’s t -test.

    Techniques Used: Activity Assay, Luciferase, Construct

    11) Product Images from "HDAC8 and STAT3 repress BMF gene activity in colon cancer cells"

    Article Title: HDAC8 and STAT3 repress BMF gene activity in colon cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.422

    Induction of p21 by MSP is not a prerequisite for apoptosis in colon cancer cells. ( a ) HCT116 cells were incubated with the transaminase inhibitor aminooxyacetic acid (AOAA) 1 h before treatment with MSP, MSC, or vehicle (control). Whole-cell lysates were immunoblotted 6 h later for histone H3 acetylation. Wedge symbol indicates increasing concentration of AOAA. ( b ) AOAA pretreatment for 1 h blocked p21 induction by MSC and the cleavage of PARP, whereas MSP was unaffected by AOAA. Open arrow, cleaved PARP. ( c ) HCT116 p21+/+ and HCT116 p21−/− cells responded similarly to MSP treatment in MTT assays; mean±S.D., n =3, * P
    Figure Legend Snippet: Induction of p21 by MSP is not a prerequisite for apoptosis in colon cancer cells. ( a ) HCT116 cells were incubated with the transaminase inhibitor aminooxyacetic acid (AOAA) 1 h before treatment with MSP, MSC, or vehicle (control). Whole-cell lysates were immunoblotted 6 h later for histone H3 acetylation. Wedge symbol indicates increasing concentration of AOAA. ( b ) AOAA pretreatment for 1 h blocked p21 induction by MSC and the cleavage of PARP, whereas MSP was unaffected by AOAA. Open arrow, cleaved PARP. ( c ) HCT116 p21+/+ and HCT116 p21−/− cells responded similarly to MSP treatment in MTT assays; mean±S.D., n =3, * P

    Techniques Used: Incubation, Concentration Assay, MTT Assay

    12) Product Images from "Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation"

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1560-y

    Proposed mechanism of action of MFX and CFX induced S-phase arrest and apoptosis. MFX and CFX causes S-phase arrest by decreasing the levels of Cyclin-A, Cyclin-E, CDK2, p21 and p27 in both the cell lines. Both FQs also leads to activation of extrinsic pathway of apoptosis via caspase-8 and ERK1/2 which then disrupts mitochondrial membrane potential via activation of Bid and proapoptotic Bak, as well as downregulates Bax and antiapoptotic protein Bcl-xL, which finally promotes activation of caspase-9,3 and leads to apoptosis. Furthermore MFX and CFX also suppresses cell survival pathway by downregulating the levels of pAKT and cMYC
    Figure Legend Snippet: Proposed mechanism of action of MFX and CFX induced S-phase arrest and apoptosis. MFX and CFX causes S-phase arrest by decreasing the levels of Cyclin-A, Cyclin-E, CDK2, p21 and p27 in both the cell lines. Both FQs also leads to activation of extrinsic pathway of apoptosis via caspase-8 and ERK1/2 which then disrupts mitochondrial membrane potential via activation of Bid and proapoptotic Bak, as well as downregulates Bax and antiapoptotic protein Bcl-xL, which finally promotes activation of caspase-9,3 and leads to apoptosis. Furthermore MFX and CFX also suppresses cell survival pathway by downregulating the levels of pAKT and cMYC

    Techniques Used: Activation Assay

    13) Product Images from "Neddylation inhibitor, MLN4924 suppresses angiogenesis in huvecs and solid cancers: in vitro and in vivo study"

    Article Title: Neddylation inhibitor, MLN4924 suppresses angiogenesis in huvecs and solid cancers: in vitro and in vivo study

    Journal: American Journal of Cancer Research

    doi:

    MLN4924 inhibits proliferation and interferes with cell cycle checkpoint regulators p21, p27 and phospho-histone H3. HUVECs were treated with DMSO as a control, or with various concentrations of MLN4924, for 24 h. A. Percentage of BrdU positive HUVECs after 24-h treatment with MLN4924 is shown. Data are presented as mean ± SD. Asterisks indicate the degree of statistical difference; *P ≤ 0.05 as compared with non-treated control. B. Cell lysates were analyzed for levels of p21, p27, and phospho-Histone-H3 (Ser10) via western blotting with specific antibodies. C. Thr-202/Tyr-204 phosphorylation of ERK1/2 and Erk in HUVECs at 24 h after exposure to MLN4924.
    Figure Legend Snippet: MLN4924 inhibits proliferation and interferes with cell cycle checkpoint regulators p21, p27 and phospho-histone H3. HUVECs were treated with DMSO as a control, or with various concentrations of MLN4924, for 24 h. A. Percentage of BrdU positive HUVECs after 24-h treatment with MLN4924 is shown. Data are presented as mean ± SD. Asterisks indicate the degree of statistical difference; *P ≤ 0.05 as compared with non-treated control. B. Cell lysates were analyzed for levels of p21, p27, and phospho-Histone-H3 (Ser10) via western blotting with specific antibodies. C. Thr-202/Tyr-204 phosphorylation of ERK1/2 and Erk in HUVECs at 24 h after exposure to MLN4924.

    Techniques Used: Western Blot

    14) Product Images from "EVI1 Inhibits Apoptosis Induced by Antileukemic Drugs via Upregulation of CDKN1A/p21/WAF in Human Myeloid Cells"

    Article Title: EVI1 Inhibits Apoptosis Induced by Antileukemic Drugs via Upregulation of CDKN1A/p21/WAF in Human Myeloid Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056308

    Regulation of the CDKN1A/p21/WAF mRNA and protein by EVI1 , etoposide, and daunorubicin. A, B) qRT-PCR on RxNA from U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide (A) or 30 nM daunorubicin (B) for 48 h. CDKN1A levels were normalized to those of the housekeeping gene B2M using the ΔΔct method [48] , with untreated U937_vec cells as a calibrator. Shown are means+SEMs from 3 independent experiments. *, p
    Figure Legend Snippet: Regulation of the CDKN1A/p21/WAF mRNA and protein by EVI1 , etoposide, and daunorubicin. A, B) qRT-PCR on RxNA from U937_vec and U937_EVI1 cells treated or not treated with 400 nM etoposide (A) or 30 nM daunorubicin (B) for 48 h. CDKN1A levels were normalized to those of the housekeeping gene B2M using the ΔΔct method [48] , with untreated U937_vec cells as a calibrator. Shown are means+SEMs from 3 independent experiments. *, p

    Techniques Used: Quantitative RT-PCR

    CDKN1A/p21/WAF overexpression partially mimics the chemotherapy resistance phenotype of EVI1 ove rexpression. A) Immunoblot analysis confirming overexpressi on of p21 in U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. β-Tubulin was used as loading control. Detection of endogenous p21 would require longer exposure times. B) Immunoblot analysis of cytoplasmatic (cyto) and nuclear (nu) extracts from U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. The same amount of protein was loaded in each lane (corresponding to up to twice as many cell equivalents for nuclear versus cytoplasmatic extracts). The cytoplasmatic protein β-tubulin and the nuclear protein RCC1 were used as loading controls. C) Cell cycle distribution of CDKN1A overexpressing and control cells. Shown are means+SEMs from 3 independent experiments. *, p
    Figure Legend Snippet: CDKN1A/p21/WAF overexpression partially mimics the chemotherapy resistance phenotype of EVI1 ove rexpression. A) Immunoblot analysis confirming overexpressi on of p21 in U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. β-Tubulin was used as loading control. Detection of endogenous p21 would require longer exposure times. B) Immunoblot analysis of cytoplasmatic (cyto) and nuclear (nu) extracts from U937_vec and U937_EVI1 cells infected with pMIA-II_CDKN1A-IRES-Ametrine. The same amount of protein was loaded in each lane (corresponding to up to twice as many cell equivalents for nuclear versus cytoplasmatic extracts). The cytoplasmatic protein β-tubulin and the nuclear protein RCC1 were used as loading controls. C) Cell cycle distribution of CDKN1A overexpressing and control cells. Shown are means+SEMs from 3 independent experiments. *, p

    Techniques Used: Over Expression, Infection

    15) Product Images from "Preclinical analysis of MTOR complex 1/2 inhibition in diffuse intrinsic pontine glioma"

    Article Title: Preclinical analysis of MTOR complex 1/2 inhibition in diffuse intrinsic pontine glioma

    Journal: Oncology Reports

    doi: 10.3892/or.2017.6122

    Drug effects on senescence and autophagy. (A) Effect of everolimus and AZD2014 on senescence, as measured by the ratio of immunofluorescence intensity of p21 compared to DAPI (error bars represent SEM). (B) Effects of everolimus and AZD2014 on senescence, as measured by the percentage of cells staining positive for β-galactosidase. (C) Effects of everolimus and AZD2014 on autophagy, as measured by autophagic flux, defined as the increase in LC3-II:tubulin western blot intensity ratio with the stated condition as compared to the stated condition plus chloroquine (an autophagy inhibitor).
    Figure Legend Snippet: Drug effects on senescence and autophagy. (A) Effect of everolimus and AZD2014 on senescence, as measured by the ratio of immunofluorescence intensity of p21 compared to DAPI (error bars represent SEM). (B) Effects of everolimus and AZD2014 on senescence, as measured by the percentage of cells staining positive for β-galactosidase. (C) Effects of everolimus and AZD2014 on autophagy, as measured by autophagic flux, defined as the increase in LC3-II:tubulin western blot intensity ratio with the stated condition as compared to the stated condition plus chloroquine (an autophagy inhibitor).

    Techniques Used: Immunofluorescence, Staining, Western Blot

    16) Product Images from "Losmapimod Overcomes Gefitinib Resistance in Non-small Cell Lung Cancer by Preventing Tetraploidization"

    Article Title: Losmapimod Overcomes Gefitinib Resistance in Non-small Cell Lung Cancer by Preventing Tetraploidization

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.01.017

    Losmapimod inhibits gefitinib-induced tetraploidization in gefitinib-resistant NSCLC cells. (A) Cell cycle analysis in untreated gefitinib-resistant NSCLC cells (left, HCC827GR and right, H1975) and in cells treated for 24 h with gefitinib or a p38 MAPK inhibitor (SB203580 or losmapimod) either alone or in combination. Tetraploid subpopulations were analyzed by the ModFit LT V4.0 software program. (B) Western blot analysis of p-YAP, p-MKK3/6 and p-p38 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 2, 6 or 10 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total YAP, MKK3/6 and p38 MAPK proteins were used as loading controls. (C) Western blot analysis of p-STAT3, p21 and cyclin D1 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 10 or 24 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total STAT3 and GAPDH proteins were used as loading controls.
    Figure Legend Snippet: Losmapimod inhibits gefitinib-induced tetraploidization in gefitinib-resistant NSCLC cells. (A) Cell cycle analysis in untreated gefitinib-resistant NSCLC cells (left, HCC827GR and right, H1975) and in cells treated for 24 h with gefitinib or a p38 MAPK inhibitor (SB203580 or losmapimod) either alone or in combination. Tetraploid subpopulations were analyzed by the ModFit LT V4.0 software program. (B) Western blot analysis of p-YAP, p-MKK3/6 and p-p38 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 2, 6 or 10 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total YAP, MKK3/6 and p38 MAPK proteins were used as loading controls. (C) Western blot analysis of p-STAT3, p21 and cyclin D1 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 10 or 24 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total STAT3 and GAPDH proteins were used as loading controls.

    Techniques Used: Cell Cycle Assay, Software, Western Blot

    17) Product Images from "Small molecule reactivation of mutant p53 to wt-like p53 through the p53-Hsp40 regulatory axis"

    Article Title: Small molecule reactivation of mutant p53 to wt-like p53 through the p53-Hsp40 regulatory axis

    Journal: Chemistry & biology

    doi: 10.1016/j.chembiol.2015.07.016

    CTM preferentially suppresses cancer cells with p53 R175H and induces p53 target genes (A) CTM shows high anticancer activity in mutant p53 R175H cells. Normal and cancer cells including p53 wild type, p53 null, mutant p53 R175H and R273H were treated with CTM for 24 hours at indicated concentrations. Cells were stained with Sulforhodamine B and measured for cell viability. Error bars represent the range of duplicates. (B) p53 target genes are highly induced in mutant p53 R175H cells. Cancer cells (R175H: CAL-33, HuCCT1, FAMPAC, KLE and TOV-112D; wild type: HCT116; null: H1299; R273H: PANC-1) with various status of p53 were treated with CTM (150 nM) for indicated times. Total RNA was extracted and subjected to quantitative real-time PCR with specific primers for p21, PUMA and MDM2. HCT116 cells were treated with etoposide (50 μM) for indicated time points as a positive control. Data shown are mean ± S.D. in triplicates and measured at the same time. (C and D) CTM-mediated p53 target protein induction in mutant p53 R175H cells. Cancer cells (R175H: CAL-33, HuCCT1, FAMPAC and KLE; R248Q: OVCAR-3; R273H: A431; wild type: HCT116; null: H1299) with various status of p53 were treated with CTM for 18 hours, and cell lysates were analyzed by western blotting with indicated antibodies. Ad-p53 was used as a positive control.
    Figure Legend Snippet: CTM preferentially suppresses cancer cells with p53 R175H and induces p53 target genes (A) CTM shows high anticancer activity in mutant p53 R175H cells. Normal and cancer cells including p53 wild type, p53 null, mutant p53 R175H and R273H were treated with CTM for 24 hours at indicated concentrations. Cells were stained with Sulforhodamine B and measured for cell viability. Error bars represent the range of duplicates. (B) p53 target genes are highly induced in mutant p53 R175H cells. Cancer cells (R175H: CAL-33, HuCCT1, FAMPAC, KLE and TOV-112D; wild type: HCT116; null: H1299; R273H: PANC-1) with various status of p53 were treated with CTM (150 nM) for indicated times. Total RNA was extracted and subjected to quantitative real-time PCR with specific primers for p21, PUMA and MDM2. HCT116 cells were treated with etoposide (50 μM) for indicated time points as a positive control. Data shown are mean ± S.D. in triplicates and measured at the same time. (C and D) CTM-mediated p53 target protein induction in mutant p53 R175H cells. Cancer cells (R175H: CAL-33, HuCCT1, FAMPAC and KLE; R248Q: OVCAR-3; R273H: A431; wild type: HCT116; null: H1299) with various status of p53 were treated with CTM for 18 hours, and cell lysates were analyzed by western blotting with indicated antibodies. Ad-p53 was used as a positive control.

    Techniques Used: Activity Assay, Mutagenesis, Staining, Real-time Polymerase Chain Reaction, Positive Control, Western Blot

    18) Product Images from "CUL4A induces epithelial-mesenchymal transition and promotes cancer metastasis by regulating ZEB1 expression"

    Article Title: CUL4A induces epithelial-mesenchymal transition and promotes cancer metastasis by regulating ZEB1 expression

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-13-2182

    CUL4A promotes proliferation of human mammary epithelial and BC cells. A, Expression levels of CUL4A, p21 and p27 by Western blotting Analysis. B–E, Cell proliferation was examined by MTT (B and D) and colony formation (C and E) assays. For colony formation assay, colonies containing more than 50 cells were counted and plotted; representative plates are shown in inserts. * P
    Figure Legend Snippet: CUL4A promotes proliferation of human mammary epithelial and BC cells. A, Expression levels of CUL4A, p21 and p27 by Western blotting Analysis. B–E, Cell proliferation was examined by MTT (B and D) and colony formation (C and E) assays. For colony formation assay, colonies containing more than 50 cells were counted and plotted; representative plates are shown in inserts. * P

    Techniques Used: Expressing, Western Blot, MTT Assay, Colony Assay

    19) Product Images from "The Chinese Herb Isolate Yuanhuacine (YHL-14) Induces G2/M Arrest in Human Cancer Cells by Up-regulating p21 Protein Expression through an p53 Protein-independent Cascade *"

    Article Title: The Chinese Herb Isolate Yuanhuacine (YHL-14) Induces G2/M Arrest in Human Cancer Cells by Up-regulating p21 Protein Expression through an p53 Protein-independent Cascade *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.513960

    Sp1 induction by YHL-14 mediates p21 transcription. A , after synchronization, cells were treated with 2 μ m YHL-14 for the indicated times. Total RNA was isolated, and RT-PCR was carried out to determine p21 mRNA levels. β-Actin mRNA levels
    Figure Legend Snippet: Sp1 induction by YHL-14 mediates p21 transcription. A , after synchronization, cells were treated with 2 μ m YHL-14 for the indicated times. Total RNA was isolated, and RT-PCR was carried out to determine p21 mRNA levels. β-Actin mRNA levels

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction

    p21 protein expression up-regulated by YHL-14 plays a key role in the induction of G 2 /M phase growth arrest in cancer cells. A and B , after synchronization, cells were treated with YHL-14 at the indicated concentrations for 12 h ( A ) or YHL-14 at 2 μ
    Figure Legend Snippet: p21 protein expression up-regulated by YHL-14 plays a key role in the induction of G 2 /M phase growth arrest in cancer cells. A and B , after synchronization, cells were treated with YHL-14 at the indicated concentrations for 12 h ( A ) or YHL-14 at 2 μ

    Techniques Used: Expressing

    20) Product Images from "Investigation of Radiation-induced Transcriptome Profile of Radioresistant Non-small Cell Lung Cancer A549 Cells Using RNA-seq"

    Article Title: Investigation of Radiation-induced Transcriptome Profile of Radioresistant Non-small Cell Lung Cancer A549 Cells Using RNA-seq

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059319

    Protein expression of candidate genes for radioresistance-associating factors in NSCLC cells. IR-altered expression of Sestrin2, TRAF4, p21, COX-2 and FDXR was assayed by western blot analysis. The results were confirmed by three independent experiments.
    Figure Legend Snippet: Protein expression of candidate genes for radioresistance-associating factors in NSCLC cells. IR-altered expression of Sestrin2, TRAF4, p21, COX-2 and FDXR was assayed by western blot analysis. The results were confirmed by three independent experiments.

    Techniques Used: Expressing, Western Blot

    21) Product Images from "Dietary restriction attenuates the accelerated aging phenotype of Sod1−/− mice"

    Article Title: Dietary restriction attenuates the accelerated aging phenotype of Sod1−/− mice

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.02.026

    Effect of DR on gene expression in the liver (n = 4). (A) Expression of p21 protein. Statistical significance (p
    Figure Legend Snippet: Effect of DR on gene expression in the liver (n = 4). (A) Expression of p21 protein. Statistical significance (p

    Techniques Used: Expressing

    22) Product Images from "Anticancer activity of Noscapine, an opioid alkaloid in combination with Cisplatin in human non-small cell lung cancer"

    Article Title: Anticancer activity of Noscapine, an opioid alkaloid in combination with Cisplatin in human non-small cell lung cancer

    Journal: Lung cancer (Amsterdam, Netherlands)

    doi: 10.1016/j.lungcan.2010.06.002

    Expression of p53, p21, caspase 3, cleaved caspase 3, PARP, cleaved PARP, Bax, Bcl 2 , survivin in H460 (A) and A549 (B) tumor cell lysates by western blotting and (C) quantitation of apoptotic protein expression. H460 cells were treated with Cis (0.8 μM),
    Figure Legend Snippet: Expression of p53, p21, caspase 3, cleaved caspase 3, PARP, cleaved PARP, Bax, Bcl 2 , survivin in H460 (A) and A549 (B) tumor cell lysates by western blotting and (C) quantitation of apoptotic protein expression. H460 cells were treated with Cis (0.8 μM),

    Techniques Used: Expressing, Western Blot, Quantitation Assay

    Expression of p53, p21, pAkt, survivin, cyclin D1, Bax, and Bcl 2 proteins in tumor lysates by western blotting (A) and (B) quantitation of apoptotic protein expression. Lane 1, untreated control tumors; lane 2, oral Nos 300 mg/kg; lane 3, Cis 2.5 mg/kg
    Figure Legend Snippet: Expression of p53, p21, pAkt, survivin, cyclin D1, Bax, and Bcl 2 proteins in tumor lysates by western blotting (A) and (B) quantitation of apoptotic protein expression. Lane 1, untreated control tumors; lane 2, oral Nos 300 mg/kg; lane 3, Cis 2.5 mg/kg

    Techniques Used: Expressing, Western Blot, Quantitation Assay

    23) Product Images from "Notch1 is a prognostic factor that is distinctly activated in the classical and proneural subtype of glioblastoma and that promotes glioma cell survival via the NF-κB(p65) pathway"

    Article Title: Notch1 is a prognostic factor that is distinctly activated in the classical and proneural subtype of glioblastoma and that promotes glioma cell survival via the NF-κB(p65) pathway

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0119-z

    Notch1 regulates the NF-κB(p65) pathway a Following transfection of U87, U251, and LN229 cells with shRNA, the expression levels of Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9 were detected by western blotting. β-Tubulin was used as a loading control. b Immunofluorescence staining showed the distribution of NF-κB(p65) in U87, U251, and LN229 cells after shRNA treatment. c Three different cell lysates were denatured and then immunoprecipitated with antibodies targeting either NICD or NF-κB(p65). Both the forward and reverse immunoprecipitation showed that NICD bound to NF-κB(p65). Whole immunoglobulin (IgG) was used as a control antibody in the immunoprecipitation assays
    Figure Legend Snippet: Notch1 regulates the NF-κB(p65) pathway a Following transfection of U87, U251, and LN229 cells with shRNA, the expression levels of Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9 were detected by western blotting. β-Tubulin was used as a loading control. b Immunofluorescence staining showed the distribution of NF-κB(p65) in U87, U251, and LN229 cells after shRNA treatment. c Three different cell lysates were denatured and then immunoprecipitated with antibodies targeting either NICD or NF-κB(p65). Both the forward and reverse immunoprecipitation showed that NICD bound to NF-κB(p65). Whole immunoglobulin (IgG) was used as a control antibody in the immunoprecipitation assays

    Techniques Used: Transfection, shRNA, Expressing, Western Blot, Immunofluorescence, Staining, Immunoprecipitation

    Effect of DAPT on NF-κB(p65) expression in glioma cells a , b DAPT-induced apoptosis of glioma cells in vitro. The percentages of apoptotic cells were significantly increased after DAPT treatment. c Immunofluorescence shows Hes1 and p65 expression in glioma cells after DAPT treatment. The scale bar corresponds to 20 µm. d After DAPT treatment, the Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 expression levels were detected by western blotting. β-Tubulin was used as a loading control. * P
    Figure Legend Snippet: Effect of DAPT on NF-κB(p65) expression in glioma cells a , b DAPT-induced apoptosis of glioma cells in vitro. The percentages of apoptotic cells were significantly increased after DAPT treatment. c Immunofluorescence shows Hes1 and p65 expression in glioma cells after DAPT treatment. The scale bar corresponds to 20 µm. d After DAPT treatment, the Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 expression levels were detected by western blotting. β-Tubulin was used as a loading control. * P

    Techniques Used: Expressing, In Vitro, Immunofluorescence, Western Blot

    24) Product Images from "Inhibition of TRF2 accelerates telomere attrition and DNA damage in naïve CD4 T cells during HCV infection"

    Article Title: Inhibition of TRF2 accelerates telomere attrition and DNA damage in naïve CD4 T cells during HCV infection

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0897-y

    T-cell senescence in HCV-infected patients versus age-matched HS. a CD28 expression on CD3 + CD4 + CD45RA + naïve and CD3 + CD4 + CD45RA - memory T cells from 11 HCV-infected patients and 7 age-matched HSs. The gating strategy and summary data of the flow cytometry analysis are shown. Each symbol represents one subject. NS non significant. b CD39 expression in T cells from HCV patients and HS. PBMCs isolated from HCV subjects and HSs were cultured in the presence of anti-CD3/CD28 for 0, 1, 3, 5 days, followed by flow cytometric analysis for CD39 expression on CD4 + T cells. Representative dot plots and summary data are shown. c CD57 expression in naïve T cells from HCV patients and HS. PBMCs were stimulated with anti-CD3/CD28 for 3 days and then analyzed for CD57 expression on CD4 + CD45RA + naïve T cells isolated from five HCV patients and five HSs. d mRNA expression of CDKN2A (p16), CDKN1A (p21), and TP53 in naïve CD4 T cells isolated from 10 HCV patients and 10 HSs. The purified CD4 + CD45RO − naïve T cells were analyzed by real-time RT-PCR for p16 ink4a , p21 cip1 , and TP53 mRNA expression. Values were normalized to GAPDH expression and are presented relative to HS. e Western blot analysis for p53 expression in naïve CD4 T cells isolated from HCV patient and HS. GAPDH serves as a loading control. Representative WB imaging and summary densitometry data are shown. f Western blot analysis for pp53, p53, p21 expressions in TCR-stimulated naïve CD4 T cells isolated from HCV patient and HS. β-Actin is used as loading control. Representative WB imaging and summary densitometry data are shown. g Measurement of ROS level in naïve CD4 T cells. Naïve CD4 T cells were isolated from HCV patients and HS and subjected to ROS measurement using the DCFDA-based Cellular ROS Detection Kit. Representative overlaid histogram and summary data for the MFI of DCFDA level in naïve CD4 T cells from HCV patients and HS are shown ( n = 4)
    Figure Legend Snippet: T-cell senescence in HCV-infected patients versus age-matched HS. a CD28 expression on CD3 + CD4 + CD45RA + naïve and CD3 + CD4 + CD45RA - memory T cells from 11 HCV-infected patients and 7 age-matched HSs. The gating strategy and summary data of the flow cytometry analysis are shown. Each symbol represents one subject. NS non significant. b CD39 expression in T cells from HCV patients and HS. PBMCs isolated from HCV subjects and HSs were cultured in the presence of anti-CD3/CD28 for 0, 1, 3, 5 days, followed by flow cytometric analysis for CD39 expression on CD4 + T cells. Representative dot plots and summary data are shown. c CD57 expression in naïve T cells from HCV patients and HS. PBMCs were stimulated with anti-CD3/CD28 for 3 days and then analyzed for CD57 expression on CD4 + CD45RA + naïve T cells isolated from five HCV patients and five HSs. d mRNA expression of CDKN2A (p16), CDKN1A (p21), and TP53 in naïve CD4 T cells isolated from 10 HCV patients and 10 HSs. The purified CD4 + CD45RO − naïve T cells were analyzed by real-time RT-PCR for p16 ink4a , p21 cip1 , and TP53 mRNA expression. Values were normalized to GAPDH expression and are presented relative to HS. e Western blot analysis for p53 expression in naïve CD4 T cells isolated from HCV patient and HS. GAPDH serves as a loading control. Representative WB imaging and summary densitometry data are shown. f Western blot analysis for pp53, p53, p21 expressions in TCR-stimulated naïve CD4 T cells isolated from HCV patient and HS. β-Actin is used as loading control. Representative WB imaging and summary densitometry data are shown. g Measurement of ROS level in naïve CD4 T cells. Naïve CD4 T cells were isolated from HCV patients and HS and subjected to ROS measurement using the DCFDA-based Cellular ROS Detection Kit. Representative overlaid histogram and summary data for the MFI of DCFDA level in naïve CD4 T cells from HCV patients and HS are shown ( n = 4)

    Techniques Used: Infection, Expressing, Flow Cytometry, Cytometry, Isolation, Cell Culture, Purification, Quantitative RT-PCR, Western Blot, Imaging

    Telomere shelterin mRNA and protein levels in T cells from HCV patients and HS. a , b mRNA expression of telomere shelterin proteins in total CD4 T cells and naïve CD4 T cells. Total CD4 T cells and CD4 + CD45RO − naïve CD4 T cells were isolated from 10 HCV patients and 10 HSs. Total RNA was isolated and analyzed by real-time RT-PCR for shelterin mRNA expression. Values were normalized to GAPDH mRNA and calculated relative to HS. c Shelterin protein expressions in naïve CD4 T cells isolated from HCV patients and HS. GAPDH is used as loading control. Representative imaging and summary data for western blot densitometry are shown ( n = 9). d TRF2 level in total CD4 T cells from HCV patients and HSs. Representative imaging and summary data for western blot are shown ( n = 4). The HCV densitometry data were first normalized to β-actin and then HS. e Naïve CD4 T cells isolated from HCV and HSs were cultured for 72 h in the presence of DMSO control or proteasome inhibitor MG132 (10 μM) for the last 4 h, followed by western blot analysis for p53, Siah-1, TRF2, p21, γH2AX expressions. β-Actin serves as loading control. f Proteasomal degradation of TRF2 through ubiquitin signaling pathway in T lymphocytes during HCV infection. Naïve CD4 T cells isolated from HCV (lanes 2 and 4) and HS (lanes 1 and 3) were lysed in immunoprecipitation (IP) buffer with 0.1% SDS. Protein concentrations were equalized and small amount of cell lysates were saved before the pull-down assay (bottom panel) and used as control. The rest of cell lysates were used for IP with TRF2 antibody or IgG control (lane 5). Immunoprecipitated complexes were pulled-down by protein A/G bead and subjected to immunoblotting with the indicated antibodies
    Figure Legend Snippet: Telomere shelterin mRNA and protein levels in T cells from HCV patients and HS. a , b mRNA expression of telomere shelterin proteins in total CD4 T cells and naïve CD4 T cells. Total CD4 T cells and CD4 + CD45RO − naïve CD4 T cells were isolated from 10 HCV patients and 10 HSs. Total RNA was isolated and analyzed by real-time RT-PCR for shelterin mRNA expression. Values were normalized to GAPDH mRNA and calculated relative to HS. c Shelterin protein expressions in naïve CD4 T cells isolated from HCV patients and HS. GAPDH is used as loading control. Representative imaging and summary data for western blot densitometry are shown ( n = 9). d TRF2 level in total CD4 T cells from HCV patients and HSs. Representative imaging and summary data for western blot are shown ( n = 4). The HCV densitometry data were first normalized to β-actin and then HS. e Naïve CD4 T cells isolated from HCV and HSs were cultured for 72 h in the presence of DMSO control or proteasome inhibitor MG132 (10 μM) for the last 4 h, followed by western blot analysis for p53, Siah-1, TRF2, p21, γH2AX expressions. β-Actin serves as loading control. f Proteasomal degradation of TRF2 through ubiquitin signaling pathway in T lymphocytes during HCV infection. Naïve CD4 T cells isolated from HCV (lanes 2 and 4) and HS (lanes 1 and 3) were lysed in immunoprecipitation (IP) buffer with 0.1% SDS. Protein concentrations were equalized and small amount of cell lysates were saved before the pull-down assay (bottom panel) and used as control. The rest of cell lysates were used for IP with TRF2 antibody or IgG control (lane 5). Immunoprecipitated complexes were pulled-down by protein A/G bead and subjected to immunoblotting with the indicated antibodies

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Imaging, Western Blot, Cell Culture, Infection, Immunoprecipitation, Pull Down Assay

    25) Product Images from "Chloroquine or Chloroquine-PI3K/Akt Pathway Inhibitor Combinations Strongly Promote ?-Irradiation-Induced Cell Death in Primary Stem-Like Glioma Cells"

    Article Title: Chloroquine or Chloroquine-PI3K/Akt Pathway Inhibitor Combinations Strongly Promote ?-Irradiation-Induced Cell Death in Primary Stem-Like Glioma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0047357

    Low doses of γIR, CQ and PI-103 synergistically induce apoptosis in GBM22 SLGCs. ( A, B ) Left: Percentage of annexin V/PI positive cells 5 or 13 d after treatment with either γIR (3.5 Gy), CQ (5 µM) or PI-103 (0.5 µM) alone, with double or triple combinations. Right: cell numbers after trypan blue staining. Lower panel in A: flow cytometry data showing that cell death occurred primarily through apoptosis. ( C ) Western blot analysis of LC3-I/II conversion and of p21 expression levels.
    Figure Legend Snippet: Low doses of γIR, CQ and PI-103 synergistically induce apoptosis in GBM22 SLGCs. ( A, B ) Left: Percentage of annexin V/PI positive cells 5 or 13 d after treatment with either γIR (3.5 Gy), CQ (5 µM) or PI-103 (0.5 µM) alone, with double or triple combinations. Right: cell numbers after trypan blue staining. Lower panel in A: flow cytometry data showing that cell death occurred primarily through apoptosis. ( C ) Western blot analysis of LC3-I/II conversion and of p21 expression levels.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Western Blot, Expressing

    26) Product Images from "From fighting depression to conquering tumors: a novel tricyclic thiazepine compound as a tubulin polymerization inhibitor"

    Article Title: From fighting depression to conquering tumors: a novel tricyclic thiazepine compound as a tubulin polymerization inhibitor

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.53

    Analyses of the cell cycle and p21 expression in H460 TaxR and H460 cells after TBPT treatment. NSCLC cells were treated with DMSO or TBPT (2 μ M) for the indicated times. ( a ) Cells were stained with PI and analyzed via flow cytometry. The two red peaks indicate the cells in the G1 and G2/M phases, respectively. The middle gray area indicates the cells in the S phase of the cell cycle. The ratio of cells in the G2/M phase is also shown. ( b ) Western blot analysis of p21 expression in NSCLC cells
    Figure Legend Snippet: Analyses of the cell cycle and p21 expression in H460 TaxR and H460 cells after TBPT treatment. NSCLC cells were treated with DMSO or TBPT (2 μ M) for the indicated times. ( a ) Cells were stained with PI and analyzed via flow cytometry. The two red peaks indicate the cells in the G1 and G2/M phases, respectively. The middle gray area indicates the cells in the S phase of the cell cycle. The ratio of cells in the G2/M phase is also shown. ( b ) Western blot analysis of p21 expression in NSCLC cells

    Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, Western Blot

    27) Product Images from "Epigallocatechin-3-gallate regulates the expression of Kruppel-like factor 4 through myocyte enhancer factor 2A"

    Article Title: Epigallocatechin-3-gallate regulates the expression of Kruppel-like factor 4 through myocyte enhancer factor 2A

    Journal: Cell Stress & Chaperones

    doi: 10.1007/s12192-013-0447-6

    EGCG regulates expression of p21, a cell cycle functional protein downstream of KLF4. Real-time PCR result revealed that EGCG increased the expression of p21 at mRNA level (* p
    Figure Legend Snippet: EGCG regulates expression of p21, a cell cycle functional protein downstream of KLF4. Real-time PCR result revealed that EGCG increased the expression of p21 at mRNA level (* p

    Techniques Used: Expressing, Functional Assay, Real-time Polymerase Chain Reaction

    28) Product Images from "TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth"

    Article Title: TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth

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

    doi: 10.1073/pnas.1420811112

    TLR4 and TP53 coregulate p21 induction. ( A C , and D ) Plot of means depicting gene expression changes in IFN-γ between tumors with gain-of-function, loss-of-function, and dominant negative TP53 mutations ( A ), and between p27 gene expression changes
    Figure Legend Snippet: TLR4 and TP53 coregulate p21 induction. ( A C , and D ) Plot of means depicting gene expression changes in IFN-γ between tumors with gain-of-function, loss-of-function, and dominant negative TP53 mutations ( A ), and between p27 gene expression changes

    Techniques Used: Expressing, Dominant Negative Mutation

    LPS*-induced p21 activation requires both TLR4 and TP53. ( A and B ) Representative photomicrographs and accompanying quantification of nuclear localized p21 in TP53 wild-type cells ( A ), as well as quantification of RNA levels ( B ) 24 h after LPS* treatment.
    Figure Legend Snippet: LPS*-induced p21 activation requires both TLR4 and TP53. ( A and B ) Representative photomicrographs and accompanying quantification of nuclear localized p21 in TP53 wild-type cells ( A ), as well as quantification of RNA levels ( B ) 24 h after LPS* treatment.

    Techniques Used: Activation Assay

    29) Product Images from "Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma"

    Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

    Journal: International journal of cancer

    doi: 10.1002/ijc.30769

    Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in ( a ) A549 and ( b ). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. ( c ) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. ( d ) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P
    Figure Legend Snippet: Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in ( a ) A549 and ( b ). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. ( c ) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. ( d ) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P

    Techniques Used: Western Blot, Flow Cytometry, Cytometry, Transfection, Labeling, Staining

    Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. ( a ) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. ( b ) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (* P
    Figure Legend Snippet: Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. ( a ) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. ( b ) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (* P

    Techniques Used: In Vitro, Migration, Western Blot, Transfection

    30) Product Images from "MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines"

    Article Title: MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.71

    Radioresistance of pmiR-210 A549 cells. ( a ) Radioresistance of wild-type A549 (A549wt) cells cultured for 2 days in normoxia (Nx) and in hypoxia 1% O 2 (Hx1%), and treated with the indicated dose of radiation. Cells were then subjected to a clonogenic cell survival assay. In x axis is the dose of X-radiation. In y axis is the surviving fraction. Mean±S.E.M. is representative of two independent experiments carried out in duplicate. ( b ) Radioresistance of both pmiR-Ctl and pmiR-210 A549 cells cultured for 2 days in normoxia (Nx) and hypoxia 1% O 2 (Hx1%). The cells were then subjected to a clonogenic cell survival assay. In x axis is the dose of X-radiation. In y axis is the surviving fraction. Mean±S.E.M. is representative of three independent experiments carried out in duplicate for a + b . ( c ) Cell morphology from microscope images of pmiR-Ctl and pmiR-210 A549 cells cultured for 2 days in normoxia (Nx) and hypoxia 1% O 2 (Hx1%), treated with 10 Gy and subject to clonogenic cell survival. ( d ) Immunoblotting of HIF-1 α , CAIX, total p53, p53-P, p21, cleaved caspase-3 (cleaved Casp.3) and β -actin in pmiR-Ctl and pmiR-210 A549 cells in normoxia (Nx) 18 h after irradiations (0, 4 and 8 Gy). ( e ) Histograms show the quantification of the p21 and the cleaved caspase-3 bands. Mean±S.E.M. is representative of three independent experiments. ** P
    Figure Legend Snippet: Radioresistance of pmiR-210 A549 cells. ( a ) Radioresistance of wild-type A549 (A549wt) cells cultured for 2 days in normoxia (Nx) and in hypoxia 1% O 2 (Hx1%), and treated with the indicated dose of radiation. Cells were then subjected to a clonogenic cell survival assay. In x axis is the dose of X-radiation. In y axis is the surviving fraction. Mean±S.E.M. is representative of two independent experiments carried out in duplicate. ( b ) Radioresistance of both pmiR-Ctl and pmiR-210 A549 cells cultured for 2 days in normoxia (Nx) and hypoxia 1% O 2 (Hx1%). The cells were then subjected to a clonogenic cell survival assay. In x axis is the dose of X-radiation. In y axis is the surviving fraction. Mean±S.E.M. is representative of three independent experiments carried out in duplicate for a + b . ( c ) Cell morphology from microscope images of pmiR-Ctl and pmiR-210 A549 cells cultured for 2 days in normoxia (Nx) and hypoxia 1% O 2 (Hx1%), treated with 10 Gy and subject to clonogenic cell survival. ( d ) Immunoblotting of HIF-1 α , CAIX, total p53, p53-P, p21, cleaved caspase-3 (cleaved Casp.3) and β -actin in pmiR-Ctl and pmiR-210 A549 cells in normoxia (Nx) 18 h after irradiations (0, 4 and 8 Gy). ( e ) Histograms show the quantification of the p21 and the cleaved caspase-3 bands. Mean±S.E.M. is representative of three independent experiments. ** P

    Techniques Used: Cell Culture, Clonogenic Cell Survival Assay, CTL Assay, Microscopy

    31) Product Images from "Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism"

    Article Title: Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21716

    D561-0775 induced cell cycle arrest in H1975 cells (A) H1975 cells were treated with D561-0775 at different concentration for 48 h. (B) Statistical analysis of cell cycle distribution after 48 h drug treatment. (C) Western blot analysis of the protein levels of p-p53 ser15 , p53, p21, Cyclin B1 and β -actin after 48 h drug treatment. (D) Statistical analysis of the densitometry signals of p-p53 ser15 , p53, p21 and Cyclin B1. All data was presented as mean ± SD (n= 3, * p
    Figure Legend Snippet: D561-0775 induced cell cycle arrest in H1975 cells (A) H1975 cells were treated with D561-0775 at different concentration for 48 h. (B) Statistical analysis of cell cycle distribution after 48 h drug treatment. (C) Western blot analysis of the protein levels of p-p53 ser15 , p53, p21, Cyclin B1 and β -actin after 48 h drug treatment. (D) Statistical analysis of the densitometry signals of p-p53 ser15 , p53, p21 and Cyclin B1. All data was presented as mean ± SD (n= 3, * p

    Techniques Used: Concentration Assay, Western Blot

    32) Product Images from "A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma"

    Article Title: A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma

    Journal: Cancer Cell International

    doi: 10.1186/s12935-017-0496-5

    Notch1 C1133Y mutation accelerates cell proliferation. a – c The CCK-8 growth curves of HN6, CAL27 and HN13 cell lines after transfections. The overexpression of wild-type Notch1 attenuated cell growth in HN6, but it enhanced cell growth in CAL27 and HN13, compared with controls. However, Notch1 C1133Y mutation accelerated cell growth in all the tested cell lines, compared with Notch1 WT -transfected cells. d The cell-cycle analysis by flow cytometry revealed a less G1 phase arrest in Notch1 C1133Y -transfected cells. e Cell-cycle-specific proteins were analyzed by western blot analysis in HN6 and HN13. Notch1 C1133Y transfection resulted in the up-regulation of CDKs (2 and 4) and cyclins (D1 and D3), whereas the expression of P27 and P21 was decreased, suggesting an expedited cell-cycle induced by Notch1 C1133Y transfection. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1 C1133Y mutation reversed the trend in all the tested cell lines. Notch1 C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection
    Figure Legend Snippet: Notch1 C1133Y mutation accelerates cell proliferation. a – c The CCK-8 growth curves of HN6, CAL27 and HN13 cell lines after transfections. The overexpression of wild-type Notch1 attenuated cell growth in HN6, but it enhanced cell growth in CAL27 and HN13, compared with controls. However, Notch1 C1133Y mutation accelerated cell growth in all the tested cell lines, compared with Notch1 WT -transfected cells. d The cell-cycle analysis by flow cytometry revealed a less G1 phase arrest in Notch1 C1133Y -transfected cells. e Cell-cycle-specific proteins were analyzed by western blot analysis in HN6 and HN13. Notch1 C1133Y transfection resulted in the up-regulation of CDKs (2 and 4) and cyclins (D1 and D3), whereas the expression of P27 and P21 was decreased, suggesting an expedited cell-cycle induced by Notch1 C1133Y transfection. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1 C1133Y mutation reversed the trend in all the tested cell lines. Notch1 C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection

    Techniques Used: Mutagenesis, CCK-8 Assay, Transfection, Over Expression, Cell Cycle Assay, Flow Cytometry, Cytometry, Western Blot, Expressing

    33) Product Images from "MiR-375 attenuates injury of cerebral ischemia/reperfusion via targetting Ctgf"

    Article Title: MiR-375 attenuates injury of cerebral ischemia/reperfusion via targetting Ctgf

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171242

    Ctfg suppressed the proliferation and migration in H/R PC12 cells ( A ) The mRNA expression of Ctgf in H/R PC12 cells transfected with miR-375 mimic and pcDNA3.0-Ctgf or pcDNA3.0-vector was determined by qRT-PCR ( B ) The effect of miR-375 mimic and pcDNA3.0-Ctgf co-transfection on H/R p12 cells proliferation was detected by EdU assay, with pcDNA3.0-vector as control. ( C ) The effect of miR-375 mimic and pcDNA3.0-Ctgf co-transfection on H/R PC12 cells’ migration was detected by transwell assay. ( D ) The effect of miR-375 mimic and pcDNA3.0-Ctgf co-transfection on protein expression of Ctgf, p21, PI3K, and p-AKT in H/R PC12 cells were determined by Western blotting. The experiments were performed in triplicate and each value represents mean ± S.D. *P
    Figure Legend Snippet: Ctfg suppressed the proliferation and migration in H/R PC12 cells ( A ) The mRNA expression of Ctgf in H/R PC12 cells transfected with miR-375 mimic and pcDNA3.0-Ctgf or pcDNA3.0-vector was determined by qRT-PCR ( B ) The effect of miR-375 mimic and pcDNA3.0-Ctgf co-transfection on H/R p12 cells proliferation was detected by EdU assay, with pcDNA3.0-vector as control. ( C ) The effect of miR-375 mimic and pcDNA3.0-Ctgf co-transfection on H/R PC12 cells’ migration was detected by transwell assay. ( D ) The effect of miR-375 mimic and pcDNA3.0-Ctgf co-transfection on protein expression of Ctgf, p21, PI3K, and p-AKT in H/R PC12 cells were determined by Western blotting. The experiments were performed in triplicate and each value represents mean ± S.D. *P

    Techniques Used: Migration, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Cotransfection, EdU Assay, Transwell Assay, Western Blot

    MiR-375 promoted the proliferation and migration, and inhibited the apoptosis in H/R PC12 cells ( A , B ) The RNA expressions of miR-375 and Ctgf (A) and the protein expression of Ctgf (B) in PC12 cells subjected to H/R or not. ( C ) The RNA expressions of miR-375 and Ctgf in H/R PC12 cells treated with NC or miR-375 . The expression of RNA was determined by qRT-PCR, and the expression of protein was determined by Western blotting. ( D ) The effect of miR-375 on H/R p12 cells proliferation was detected by CCK-8 assay. ( E ) The effect of miR-375 on H/R p12 cells apoptosis was examined by Hoechst 33258 staining. ( F ) The expressions of Ctgf in H/R PC12 cells treated with NC or miR-375 was detected by immunofluorescence staining. ( G , H ) The effect of miR-375 on H/R p12 cell migration was detected by transwell assay, and the migrant cells were calculated. ( I ) The effect of miR-375 on protein expression of Ctgf, p21, PI3K, and p-AKT in H/R p12 cells was examined by Western blotting. The experiments were performed in triplicate and each value represents mean ± S.D. *** P
    Figure Legend Snippet: MiR-375 promoted the proliferation and migration, and inhibited the apoptosis in H/R PC12 cells ( A , B ) The RNA expressions of miR-375 and Ctgf (A) and the protein expression of Ctgf (B) in PC12 cells subjected to H/R or not. ( C ) The RNA expressions of miR-375 and Ctgf in H/R PC12 cells treated with NC or miR-375 . The expression of RNA was determined by qRT-PCR, and the expression of protein was determined by Western blotting. ( D ) The effect of miR-375 on H/R p12 cells proliferation was detected by CCK-8 assay. ( E ) The effect of miR-375 on H/R p12 cells apoptosis was examined by Hoechst 33258 staining. ( F ) The expressions of Ctgf in H/R PC12 cells treated with NC or miR-375 was detected by immunofluorescence staining. ( G , H ) The effect of miR-375 on H/R p12 cell migration was detected by transwell assay, and the migrant cells were calculated. ( I ) The effect of miR-375 on protein expression of Ctgf, p21, PI3K, and p-AKT in H/R p12 cells was examined by Western blotting. The experiments were performed in triplicate and each value represents mean ± S.D. *** P

    Techniques Used: Migration, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Staining, Immunofluorescence, Transwell Assay

    34) Product Images from "Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma"

    Article Title: Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma

    Journal: Molecular biology reports

    doi: 10.1007/s11033-008-9378-1

    Western blots demonstrate Runx2 is highly expressed in OS1 but weakly expressed in HFOB. Pocket protein pRB was detected in OS1, but the growth suppressor p53 and the CDK inhibitor p21, which is a downstream target of p53, is not detected in OS1 cells.
    Figure Legend Snippet: Western blots demonstrate Runx2 is highly expressed in OS1 but weakly expressed in HFOB. Pocket protein pRB was detected in OS1, but the growth suppressor p53 and the CDK inhibitor p21, which is a downstream target of p53, is not detected in OS1 cells.

    Techniques Used: Western Blot

    35) Product Images from "P53 suppresses ribonucleotide reductase via inhibiting mTORC1"

    Article Title: P53 suppresses ribonucleotide reductase via inhibiting mTORC1

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17440

    Inhibition of HDM2 by nutlin-3 decreases RRM1 and RRM2 in cancer cells with wild type TP53 (A) Rh18 cells were treated with different concentrations of nutlin-3 as indicated for 24 hr. Total proteins were extracted for immunoblotting of RRM1, RRM2, γH2AX, p21, p53R2, p53, S6, pS6-235/6 and 4E-BP1. (B) Rh30 cells were treated with different concentrations of nutlin-3 as indicated for 24 hr. Total proteins were extracted for immunoblotting of RRM1, RRM2, γH2AX, p21, p53R2, p53, S6, pS6-235/6 and 4E-BP1. GAPDH and Actin served as loading controls.
    Figure Legend Snippet: Inhibition of HDM2 by nutlin-3 decreases RRM1 and RRM2 in cancer cells with wild type TP53 (A) Rh18 cells were treated with different concentrations of nutlin-3 as indicated for 24 hr. Total proteins were extracted for immunoblotting of RRM1, RRM2, γH2AX, p21, p53R2, p53, S6, pS6-235/6 and 4E-BP1. (B) Rh30 cells were treated with different concentrations of nutlin-3 as indicated for 24 hr. Total proteins were extracted for immunoblotting of RRM1, RRM2, γH2AX, p21, p53R2, p53, S6, pS6-235/6 and 4E-BP1. GAPDH and Actin served as loading controls.

    Techniques Used: Inhibition

    36) Product Images from "Enhancement of Proliferation and Invasion of Gastric Cancer Cell by KDM5C Via Decrease in p53 Expression"

    Article Title: Enhancement of Proliferation and Invasion of Gastric Cancer Cell by KDM5C Via Decrease in p53 Expression

    Journal: Technology in Cancer Research & Treatment

    doi: 10.1177/1533034616629261

    The effects of KDM5C on the p53. A, The expression of p53, p27, and p21 in KDM5C-transfected cells was examined using Western blotting. β-Actin was used as a loading control. B, The transfection efficiency of p53 siRNA 24 hours after transfection was measured using Western blot analyses. β-Actin was used as a loading control. C, The graphs show the proliferation ability of KDM5C knockdown MKN45 and its control cells after the cells had been pretreated with p53 siRNA and its negative control. D, The summary graphs show the migration ability of KDM5C knockdown MKN45 and its control cells after the cells had been transfected with p53 siRNA and its negative control. The data represent the mean number of cells per field and are presented as the means ± SD ** P
    Figure Legend Snippet: The effects of KDM5C on the p53. A, The expression of p53, p27, and p21 in KDM5C-transfected cells was examined using Western blotting. β-Actin was used as a loading control. B, The transfection efficiency of p53 siRNA 24 hours after transfection was measured using Western blot analyses. β-Actin was used as a loading control. C, The graphs show the proliferation ability of KDM5C knockdown MKN45 and its control cells after the cells had been pretreated with p53 siRNA and its negative control. D, The summary graphs show the migration ability of KDM5C knockdown MKN45 and its control cells after the cells had been transfected with p53 siRNA and its negative control. The data represent the mean number of cells per field and are presented as the means ± SD ** P

    Techniques Used: Expressing, Transfection, Western Blot, Negative Control, Migration

    37) Product Images from "Chemosensitivity enhancement toward arsenic trioxide by inhibition of histone deacetylase in NB4 cell line"

    Article Title: Chemosensitivity enhancement toward arsenic trioxide by inhibition of histone deacetylase in NB4 cell line

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060516646238

    Western blot analysis of protein levels in NB4 cells treated with vorinostat with or without arsenic trioxide (ATO) for 48 h. The proteins detected were as follows: promyelocytic leukaemia (PML)/retinoic acid receptor alpha (RAR-α) fusion protein, Bcl-2, p21, acetyl-histone H3, acetyl-histone H4, Akt and pAkt. β-actin was used as an internal loading control. C: blank control; S: 0.5 µmol/l vorinostat; A: 2 µmol/l ATO; S + A: 0.5 µmol/l vorinostat + 2 µmol/l ATO.
    Figure Legend Snippet: Western blot analysis of protein levels in NB4 cells treated with vorinostat with or without arsenic trioxide (ATO) for 48 h. The proteins detected were as follows: promyelocytic leukaemia (PML)/retinoic acid receptor alpha (RAR-α) fusion protein, Bcl-2, p21, acetyl-histone H3, acetyl-histone H4, Akt and pAkt. β-actin was used as an internal loading control. C: blank control; S: 0.5 µmol/l vorinostat; A: 2 µmol/l ATO; S + A: 0.5 µmol/l vorinostat + 2 µmol/l ATO.

    Techniques Used: Western Blot

    38) Product Images from "Dabrafenib inhibits the growth of BRAF‐WT cancers through CDK16 and NEK9 inhibition"

    Article Title: Dabrafenib inhibits the growth of BRAF‐WT cancers through CDK16 and NEK9 inhibition

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12152

    Effect of NEK9 silencing on growth of RAS ‐mutant cancer cell lines. (A) siRNA knockdown of NEK9 reduces the growth of BRAF ‐ and NRAS ‐mutant melanoma cell lines. Cells were transfected with siRNA #1 or #2 overnight before quantification of cell numbers by trypan blue. (B) NEK9 silencing (siRNA #1) leads to G1‐phase cell cycle arrest in 1205Lu and WM1366 cells. (C) Knockdown of NEK9 increases p21 expression and inhibits phosphorylation of CHK1. Cells were transfected with siRNA overnight prior to western blotting for NEK9, p21, pCHK1, CHK1, and GAPDH. Numbers indicate expression relative to control. (D) Dabrafenib, but not vemurafenib, inhibits pCHK1 and increases p21 expression in WM1366 and MIA PaCa‐2 cells. Cells were treated with dabrafenib (100 n m ), vemurafenib (1 μ m ), or vehicle for 24 h (MIA PaCa‐2) or 48 h (WM1366) and subjected to western blot for p‐CHK1, CHK1, p21, and GAPDH. Numbers indicate expression relative to control. (E) Left panel: Dabrafenib selectively inhibits pCHK1 and increases p21 expression in p53−/− cells. HCT116 p53−/− and p53+/+ cells were treated with dabrafenib (100 n m ), vemurafenib (1 μ m ), or vehicle for 48 h and subjected to western blot for p‐CHK1, CHK1, p21, and GAPDH. Right panel: effect of vemurafenib (1 μ m ) and dabrafenib (100 n m ) on proliferation of HCT116 p53−/− and p53+/+ cells. * indicates significant difference from controls ( P
    Figure Legend Snippet: Effect of NEK9 silencing on growth of RAS ‐mutant cancer cell lines. (A) siRNA knockdown of NEK9 reduces the growth of BRAF ‐ and NRAS ‐mutant melanoma cell lines. Cells were transfected with siRNA #1 or #2 overnight before quantification of cell numbers by trypan blue. (B) NEK9 silencing (siRNA #1) leads to G1‐phase cell cycle arrest in 1205Lu and WM1366 cells. (C) Knockdown of NEK9 increases p21 expression and inhibits phosphorylation of CHK1. Cells were transfected with siRNA overnight prior to western blotting for NEK9, p21, pCHK1, CHK1, and GAPDH. Numbers indicate expression relative to control. (D) Dabrafenib, but not vemurafenib, inhibits pCHK1 and increases p21 expression in WM1366 and MIA PaCa‐2 cells. Cells were treated with dabrafenib (100 n m ), vemurafenib (1 μ m ), or vehicle for 24 h (MIA PaCa‐2) or 48 h (WM1366) and subjected to western blot for p‐CHK1, CHK1, p21, and GAPDH. Numbers indicate expression relative to control. (E) Left panel: Dabrafenib selectively inhibits pCHK1 and increases p21 expression in p53−/− cells. HCT116 p53−/− and p53+/+ cells were treated with dabrafenib (100 n m ), vemurafenib (1 μ m ), or vehicle for 48 h and subjected to western blot for p‐CHK1, CHK1, p21, and GAPDH. Right panel: effect of vemurafenib (1 μ m ) and dabrafenib (100 n m ) on proliferation of HCT116 p53−/− and p53+/+ cells. * indicates significant difference from controls ( P

    Techniques Used: Mutagenesis, Transfection, Expressing, Western Blot

    39) Product Images from "A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells"

    Article Title: A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25138

    Differential expression of p53 responsive pathway proteins (p21, Bcl2 and SOD2) in response to the treatment of PhiKan083 and SCH529074 in combination with Dox and TMZ (C: Control, P: PhiKan083-50 μM, S: SCH529074-1. 25 μM, D: Dox-1 μM, T: TMZ-100 μM, P+D: PhiKan083-50 μM and Dox-1 μM, P+T: PhiKan083-50 μM and TMZ-100 μM, S+D: SCH529074-1.25 μM and Dox-1 μM, S+T: SCH529074-1.25 μM and TMZ-100 μM)
    Figure Legend Snippet: Differential expression of p53 responsive pathway proteins (p21, Bcl2 and SOD2) in response to the treatment of PhiKan083 and SCH529074 in combination with Dox and TMZ (C: Control, P: PhiKan083-50 μM, S: SCH529074-1. 25 μM, D: Dox-1 μM, T: TMZ-100 μM, P+D: PhiKan083-50 μM and Dox-1 μM, P+T: PhiKan083-50 μM and TMZ-100 μM, S+D: SCH529074-1.25 μM and Dox-1 μM, S+T: SCH529074-1.25 μM and TMZ-100 μM)

    Techniques Used: Expressing

    40) Product Images from "Upregulation of cell cycle genes in head and neck cancer patients may be antagonized by erufosine’s down regulation of cell cycle processes in OSCC cells"

    Article Title: Upregulation of cell cycle genes in head and neck cancer patients may be antagonized by erufosine’s down regulation of cell cycle processes in OSCC cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23537

    ( A ) Cell cycle analysis of HN-5 and SCC-61 post 24 h of erufosine treatment. Both cell lines were treated with IC50 concentration of the drug and analyzed by PI staining. Both cell lines show a G2/M block. Cell fractions (%) from different phases of the cell cycle are given in the table below the figure. Ten thousand cells (events) per sample were analyzed to study distribution of the cells. ( B ) The protein expression of p21 and p27 in response to erufosine exposure in HN-5 and SCC-61 cells. An increase in expression was seen in both p21 and p27 levels. ( C ) Protein expression of Cdc25C and p-Cdc25C Thr48 in response to erufosine exposure post 24 h in HN-5 and SCC-61 cells. A dose dependent decrease in protein levels was observed. Protein level changes were deduced by dividing the densitometry output for each band by that for the corresponding β-actin band. ( D ) Inhibition of colony formation by erufosine: Significant anti-clonogenic effects were observed in HN-5 and SCC-61 cells after exposure to erufosine at IC20 concentrations. Asterisks denote significant differences ( P
    Figure Legend Snippet: ( A ) Cell cycle analysis of HN-5 and SCC-61 post 24 h of erufosine treatment. Both cell lines were treated with IC50 concentration of the drug and analyzed by PI staining. Both cell lines show a G2/M block. Cell fractions (%) from different phases of the cell cycle are given in the table below the figure. Ten thousand cells (events) per sample were analyzed to study distribution of the cells. ( B ) The protein expression of p21 and p27 in response to erufosine exposure in HN-5 and SCC-61 cells. An increase in expression was seen in both p21 and p27 levels. ( C ) Protein expression of Cdc25C and p-Cdc25C Thr48 in response to erufosine exposure post 24 h in HN-5 and SCC-61 cells. A dose dependent decrease in protein levels was observed. Protein level changes were deduced by dividing the densitometry output for each band by that for the corresponding β-actin band. ( D ) Inhibition of colony formation by erufosine: Significant anti-clonogenic effects were observed in HN-5 and SCC-61 cells after exposure to erufosine at IC20 concentrations. Asterisks denote significant differences ( P

    Techniques Used: Cell Cycle Assay, Concentration Assay, Staining, Blocking Assay, Expressing, Inhibition

    Related Articles

    Expressing:

    Article Title: GOLPH3 predicts survival of colorectal cancer patients treated with 5-fluorouracil-based adjuvant chemotherapy
    Article Snippet: .. The expression of proteins was detected using primary antibody against GOLPH3 (Cat#: 19112-1-AP; 1:500; Proteintech), PARP (Cat#: 9542; 1:1000; Cell Signaling Technology), and β-actin (Cat#: TA-09; 1:1000; ZSGB-BIO). .. The signal was detected by the ECL Western blot detection kit (Amersham, Little Chalfont, UK).

    other:

    Article Title: Capsaicin-induced TRIB3 upregulation promotes apoptosis in cancer cells
    Article Snippet: The antibodies against PARP, caspase 3, caspase 8, caspase 9, caspase 12, phospho-p38, phospho-ERK1/2, phospho-JNK, and phosphor-AKT were products of Cell Signaling Technology (Beverly, MA, USA).

    Article Title: Beclin 1 acetylation impairs the anticancer effect of aspirin in colorectal cancer cells
    Article Snippet: Antibodies and reagents Anti-Beclin 1, acetylated lysine, PARP, p-AMPKα (T172), p-ACC (S79) and p-S6K1 (T389) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

    SDS Page:

    Article Title: Homoharringtonine, a clinically approved anti-leukemia drug, sensitizes tumor cells for TRAIL-induced necroptosis
    Article Snippet: .. Proteins (20 μg per lane) were separated by SDS-PAGE, transferred onto nitrocellulose membranes and reactive proteins were detected with antibodies against RIPK1 (610459, BD Biosciences), RIPK3 (PAB0287, Abnova, Heidelberg, Germany), MLKL (2103620, Sigma-Aldrich), pMLKL (phospho S358, ab187091, Abcam, Cambridge, UK), PARP-1 (9542, Cell Signaling, Danvers, USA), caspase-8 (1C12, 9746, Cell Signaling), cleaved caspase-3 (9661, Cell Signaling) or actin (A1978, Sigma-Aldrich) by chemiluminescence (LumiGLO, Cell Signaling). .. RNA interference The siRNAs specific for human RIPK1 (D-004445-03), RIPK3 (D-003534-01) as well as the negative control siRNA (#1, D-001206-13) were obtained from Thermo Scientific (Schwerte, Germany).

    Mass Spectrometry:

    Article Title: TIMELESS Forms a Complex with PARP1 Distinct from Its Complex with TIPIN and Plays a Role in the DNA Damage Response
    Article Snippet: .. Antibodies The following antibodies were used: anti-TIMELESS (Bethyl Laboratories, A300-961A and A300-960A, the latter used for IP), anti-PARP1 (Invitrogen, 436400), anti-PARP1 (Cell Signaling Technology, 9542 and 46D11, the latter used for IF), anti-FLAG M2 (Sigma, F3165), anti-p-Chk1 (Cell Signaling, 2344), anti-p-Chk2 (Cell Signaling, 2661), anti-p-ATM (Cell Signaling, 4526), anti-LIG3 (Bethyl, A301-637A), anti-HLTF (Bethyl, A300-229A), anti-KU70 (Santa Cruz Biotechnology, sc-9033), anti-KU80 (Neomarkers, MS-285-P1; Cell Signaling, 2180), anti-XRCC1 (Cell Signaling, 2735), anti-DNA-PK (Santa Cruz, sc-5282), anti-DNA-PK (Cell Signaling, 12311), anti-TIPIN (Bethyl, A301-474A-1), anti-RPA1 (Santa Cruz, sc46504), anti-RPA2 (Millipore, 04-1481), anti-SSRP1 (Abcam, ab26212), anti-SPT16 (Cell Signaling, 12191), anti-SKP1 (Santa Cruz, sc-5281), and anti-phospho-H2AX (Millipore, 05-636). ..

    Protein Concentration:

    Article Title: Inhibition of Wnt3a/FOXM1/β-Catenin Axis and Activation of GSK3β and Caspases are Critically Involved in Apoptotic Effect of Moracin D in Breast Cancers
    Article Snippet: .. Then, the supernatants were quantified for protein concentration by using protein quantified assay kit (Bio-Rad, Hercules, CA, USA), The proteins lysate samples were separated on 10% Tris gels and transferred to a ECL transfer membrane for detection with antibodies for PARP, caspase 3, caspase 7, FOXM1, Wnt3a, β-catenin, Cyclin D1, c-Myc (Cell signaling Technology, Beverly, MA, USA), phospho-GSK3β (Tyr 216) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and β-actin (Sigma, St. Louis, MO, USA). .. Co-Immunoprecipitation MDA-MB-231 cells were lyzed in lysis buffer and then were immuneprecipitated with FOXM1 antibody or normal immunoglobulin G antibody.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Cell Signaling Technology Inc p21
    Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors <t>(p21,</t> 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.
    P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 544 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p21/product/Cell Signaling Technology Inc
    Average 94 stars, based on 544 article reviews
    Price from $9.99 to $1999.99
    p21 - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc anti p21
    NCL-6/S*A expression results in an increased expression of apoptotic markers downstream the p53 pathway. (A) Western blot analyses for cells grown without Dx for the indicated period indicating inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A expression resulted in an increase in p53 and <t>p21</t> protein levels. Increased expression of BH3-only pro-apoptotic markers (BID, BIM and PUMA) were observed as early as 1 d or 4 d of induced NCL-6/S*A expression. (B) Plots of p53, p21 and BH3-only protein levels shown in 6A. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 1) cells. The graph is representative of at least two independent experiments.
    Anti P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p21/product/Cell Signaling Technology Inc
    Average 94 stars, based on 315 article reviews
    Price from $9.99 to $1999.99
    anti p21 - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    89
    Cell Signaling Technology Inc monoclonal antibodies against p21
    Evaluation of 1α,25(OH) 2 D 3 and MART-10 effect on <t>p21</t> and p27 expressions in SNU308 cells. After two days of treatment (10 −6 to 10 −8 1α,25(OH) 2 D 3 or 10 −7 to 10 −9 M MART-10), p21 and p27 expressions were determined by western blot. ( a ) A western blot showing p21 and p27 expression in SNU308 cells with or without treatment. Actin was used as the loading control. (cropped). ( b ) Quantitative result of western blot. Both 1α,25(OH) 2 D 3 and MART-10 increased p27 expression in SNU308 cells without obvious effect on p21. MART-10 was more effective than 1α,25(OH) 2 D 3 . Each value is a mean ± SD of three to five determinations. *p
    Monoclonal Antibodies Against P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal antibodies against p21/product/Cell Signaling Technology Inc
    Average 89 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    monoclonal antibodies against p21 - by Bioz Stars, 2020-10
    89/100 stars
      Buy from Supplier

    85
    Cell Signaling Technology Inc anti human p21 ab
    hUHRF1 suppresses <t>p21</t> expression in cooperation with G9a. ( A ) Quantitative RT–PCR analysis of p21 expression. Total RNA was isolated from HeLa cells transfected with siRNAs as indicated. The Q-PCR data normalized by GAPDH control are shown as the means ± SD of triplicate determinations from four independent experiments. Statistical significance of the differences among the groups was determined by Student's t -test. * P
    Anti Human P21 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human p21 ab/product/Cell Signaling Technology Inc
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human p21 ab - by Bioz Stars, 2020-10
    85/100 stars
      Buy from Supplier

    Image Search Results


    Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors (p21, 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.

    Journal: PLoS ONE

    Article Title: Epstein-Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells

    doi: 10.1371/journal.pone.0017809

    Figure Lengend Snippet: Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors (p21, 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.

    Article Snippet: All other antibodies were commercially available: KSHV K8.1 (ABI, Columbia, MD); CDK6, pRb/S807/S811 and p21 (Cell Signaling Technology, Danvers, MA); c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA); 14-3-3σ (GeneTex, Irvine, CA); CCND2 (BD Pharmingen, Franklin Lakes, NJ); α-tubulin (Millipore, Billerica, MA); β-actin and M2-FLAG (Sigma-Aldrich, St. Louis, MO).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Expressing

    NCL-6/S*A expression results in an increased expression of apoptotic markers downstream the p53 pathway. (A) Western blot analyses for cells grown without Dx for the indicated period indicating inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A expression resulted in an increase in p53 and p21 protein levels. Increased expression of BH3-only pro-apoptotic markers (BID, BIM and PUMA) were observed as early as 1 d or 4 d of induced NCL-6/S*A expression. (B) Plots of p53, p21 and BH3-only protein levels shown in 6A. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 1) cells. The graph is representative of at least two independent experiments.

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: NCL-6/S*A expression results in an increased expression of apoptotic markers downstream the p53 pathway. (A) Western blot analyses for cells grown without Dx for the indicated period indicating inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A expression resulted in an increase in p53 and p21 protein levels. Increased expression of BH3-only pro-apoptotic markers (BID, BIM and PUMA) were observed as early as 1 d or 4 d of induced NCL-6/S*A expression. (B) Plots of p53, p21 and BH3-only protein levels shown in 6A. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 1) cells. The graph is representative of at least two independent experiments.

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Expressing, Western Blot, Software

    NCL-WT and 6/S*A interact with endogenous NCL. Nuclear extracts (NE) were prepared from cells grown without Dx for at least 10 d for NCL-WT or 6/S*A expression. Ctrl represents control cells without exogenous NCL expression. Equal amounts of NE protein from these cells were then subjected to co-immunoprecipitation using anti-FLAG M2 beads. Western analyses of NE and bound fractions were analyzed by anti-NCL (to detect: -exogenous 3xFlag-tagged NCL, upper band and –endogenous NCL, lower band), anti-Flag, anti-p53 and anti-p21. Anti-TOPOII β blot serves as the loading control for NE. The data is representative of three independent experiments performed with 10 d–20 d of WT or 6/S*A expression. This data supports the hypothesis that NCL (both WT and 6/S*A) can associate with endogenous NCL (i.e. NCL-NCL interactions).

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: NCL-WT and 6/S*A interact with endogenous NCL. Nuclear extracts (NE) were prepared from cells grown without Dx for at least 10 d for NCL-WT or 6/S*A expression. Ctrl represents control cells without exogenous NCL expression. Equal amounts of NE protein from these cells were then subjected to co-immunoprecipitation using anti-FLAG M2 beads. Western analyses of NE and bound fractions were analyzed by anti-NCL (to detect: -exogenous 3xFlag-tagged NCL, upper band and –endogenous NCL, lower band), anti-Flag, anti-p53 and anti-p21. Anti-TOPOII β blot serves as the loading control for NE. The data is representative of three independent experiments performed with 10 d–20 d of WT or 6/S*A expression. This data supports the hypothesis that NCL (both WT and 6/S*A) can associate with endogenous NCL (i.e. NCL-NCL interactions).

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Expressing, Immunoprecipitation, Western Blot

    NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression. (A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression. (A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Clone Assay, Expressing, Western Blot, Derivative Assay, Software

    Mechanistic model by which nucleolin phosphorylation by CK2 regulates cell proliferation. NCL-WT and phosphorylation-deficient mutant (6/S*A) activate p53 checkpoint and increase corresponding p21 levels. However, NCL-WT expression allows cells through S-phase progression and resumes cell cycle. On the other hand, NCL-6/S*A acts as a dominant-negative mutant that negates NCL-WT functions possibly through oligomerization causing inhibition of cell proliferation and initiating apoptotic pathway.

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: Mechanistic model by which nucleolin phosphorylation by CK2 regulates cell proliferation. NCL-WT and phosphorylation-deficient mutant (6/S*A) activate p53 checkpoint and increase corresponding p21 levels. However, NCL-WT expression allows cells through S-phase progression and resumes cell cycle. On the other hand, NCL-6/S*A acts as a dominant-negative mutant that negates NCL-WT functions possibly through oligomerization causing inhibition of cell proliferation and initiating apoptotic pathway.

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Mutagenesis, Expressing, Dominant Negative Mutation, Inhibition

    Evaluation of 1α,25(OH) 2 D 3 and MART-10 effect on p21 and p27 expressions in SNU308 cells. After two days of treatment (10 −6 to 10 −8 1α,25(OH) 2 D 3 or 10 −7 to 10 −9 M MART-10), p21 and p27 expressions were determined by western blot. ( a ) A western blot showing p21 and p27 expression in SNU308 cells with or without treatment. Actin was used as the loading control. (cropped). ( b ) Quantitative result of western blot. Both 1α,25(OH) 2 D 3 and MART-10 increased p27 expression in SNU308 cells without obvious effect on p21. MART-10 was more effective than 1α,25(OH) 2 D 3 . Each value is a mean ± SD of three to five determinations. *p

    Journal: Scientific Reports

    Article Title: MART-10 represses cholangiocarcinoma cell growth and high vitamin D receptor expression indicates better prognosis for cholangiocarcinoma

    doi: 10.1038/srep43773

    Figure Lengend Snippet: Evaluation of 1α,25(OH) 2 D 3 and MART-10 effect on p21 and p27 expressions in SNU308 cells. After two days of treatment (10 −6 to 10 −8 1α,25(OH) 2 D 3 or 10 −7 to 10 −9 M MART-10), p21 and p27 expressions were determined by western blot. ( a ) A western blot showing p21 and p27 expression in SNU308 cells with or without treatment. Actin was used as the loading control. (cropped). ( b ) Quantitative result of western blot. Both 1α,25(OH) 2 D 3 and MART-10 increased p27 expression in SNU308 cells without obvious effect on p21. MART-10 was more effective than 1α,25(OH) 2 D 3 . Each value is a mean ± SD of three to five determinations. *p

    Article Snippet: The primary antibodies used in this study were monoclonal antibodies against p21 (#2946, Cell Signal, Beverly, MA, USA,), p27 (#3698, Cell Signal), CDK4 (cell signal, #2906, Beverly, MA, USA), CDK6 (cell signal, #3136, Beverly, MA, USA), cyclin D3 (cell signal, #2936, Beverly, MA, USA), NGAL(#PAB9543, Abnova Corporation, Taipei, Taiwan).

    Techniques: Western Blot, Expressing

    hUHRF1 suppresses p21 expression in cooperation with G9a. ( A ) Quantitative RT–PCR analysis of p21 expression. Total RNA was isolated from HeLa cells transfected with siRNAs as indicated. The Q-PCR data normalized by GAPDH control are shown as the means ± SD of triplicate determinations from four independent experiments. Statistical significance of the differences among the groups was determined by Student's t -test. * P

    Journal: Nucleic Acids Research

    Article Title: UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells

    doi: 10.1093/nar/gkn961

    Figure Lengend Snippet: hUHRF1 suppresses p21 expression in cooperation with G9a. ( A ) Quantitative RT–PCR analysis of p21 expression. Total RNA was isolated from HeLa cells transfected with siRNAs as indicated. The Q-PCR data normalized by GAPDH control are shown as the means ± SD of triplicate determinations from four independent experiments. Statistical significance of the differences among the groups was determined by Student's t -test. * P

    Article Snippet: Antibodies (Ab) used for immunoprecipitation and western analyses were as follows: anti-GFP Ab (Roche Applied Science), anti-hUHRF1 Ab (BD Biosciences), anti-G9a Ab (Sigma), anti-human p21 Ab (Cell Signaling Technology), anti-mouse p21 Ab (Abcam), anti-dimethyl histone H3 (Lys9) Ab (Millipore), anti-histone H3 Ab (Cell Signaling Technology), anti-phospho-histone H3 (Ser10) Ab (Cell Signaling Technology), anti-actin Ab (Sigma) and anti-DNMT1 Ab (New England Biolabs).

    Techniques: Expressing, Quantitative RT-PCR, Isolation, Transfection, Polymerase Chain Reaction

    hUHRF1 recruits G9a and other chromatin modification enzymes to p21 promoter. ( A ) Linearity of PCR amplification using primer sets for proximal (−385 to −240) and distal (−4164 to −3959) regions of p21 promoter with increasing amount of input DNA. ( B ) ChIP analysis of p21 promoter after KD of hUHRF1. HeLa cells were transfected with either control siRNA (CTL KD) or hUHRF1 siRNA (hUHRF1 KD). Using the chromatin isolated from the KD cells, ChIP was performed to detect the proteins or histone modification as indicated at the top of the panel. 5% input is shown. ( C ) Quantitative ChIP analysis for relative p21 promoter occupancy of hUHRF1, G9a and dimethylated H3K9 (H3K9me2) after KD of hUHRF1 or G9a. Q-PCR data of each group were normalized to its input as % input. The relative p21 promoter occupancy of hUHRF1 or G9a KD samples represents the fold change in percentage input over that of the CTL KD. Error bars indicate standard deviation of three independent experiments. ( D ) ChIP analysis of p21 promoter after KD of DNMT1. HeLa cells were transfected with either control siRNA (CTL KD) or DNMT1 siRNA (DNMT1 KD). CHIP was performed as described in (B) and 5% input is shown.

    Journal: Nucleic Acids Research

    Article Title: UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells

    doi: 10.1093/nar/gkn961

    Figure Lengend Snippet: hUHRF1 recruits G9a and other chromatin modification enzymes to p21 promoter. ( A ) Linearity of PCR amplification using primer sets for proximal (−385 to −240) and distal (−4164 to −3959) regions of p21 promoter with increasing amount of input DNA. ( B ) ChIP analysis of p21 promoter after KD of hUHRF1. HeLa cells were transfected with either control siRNA (CTL KD) or hUHRF1 siRNA (hUHRF1 KD). Using the chromatin isolated from the KD cells, ChIP was performed to detect the proteins or histone modification as indicated at the top of the panel. 5% input is shown. ( C ) Quantitative ChIP analysis for relative p21 promoter occupancy of hUHRF1, G9a and dimethylated H3K9 (H3K9me2) after KD of hUHRF1 or G9a. Q-PCR data of each group were normalized to its input as % input. The relative p21 promoter occupancy of hUHRF1 or G9a KD samples represents the fold change in percentage input over that of the CTL KD. Error bars indicate standard deviation of three independent experiments. ( D ) ChIP analysis of p21 promoter after KD of DNMT1. HeLa cells were transfected with either control siRNA (CTL KD) or DNMT1 siRNA (DNMT1 KD). CHIP was performed as described in (B) and 5% input is shown.

    Article Snippet: Antibodies (Ab) used for immunoprecipitation and western analyses were as follows: anti-GFP Ab (Roche Applied Science), anti-hUHRF1 Ab (BD Biosciences), anti-G9a Ab (Sigma), anti-human p21 Ab (Cell Signaling Technology), anti-mouse p21 Ab (Abcam), anti-dimethyl histone H3 (Lys9) Ab (Millipore), anti-histone H3 Ab (Cell Signaling Technology), anti-phospho-histone H3 (Ser10) Ab (Cell Signaling Technology), anti-actin Ab (Sigma) and anti-DNMT1 Ab (New England Biolabs).

    Techniques: Modification, Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Transfection, CTL Assay, Isolation, Standard Deviation

    hUHRF1 cooperates with G9a to enhance the transcriptional repression of p21 promoter. ( A ) hUHRF1-mediated transcriptional repression of the p21 promoter-luciferase reporter. COS-7 cells were cotransfected with the reporter (2 μg) and increasing amounts of EGFP-hUHRF1 as indicated at the bottom of the panel. ( B ) Enhanced transcriptional repression by G9a in the presence of exogenous hUHRF1. Luciferase activities were measured from COS-7 cells cotransfected with the same reporter described earlier and increasing amounts of EGFP-G9a (0.1–1 μg) with or without a constant amount (0.4 μg) of EGFP-hUHRF1. The luciferase assays were performed as described in Figure 4, and the data represent the means ± SD of duplicate determinations from three separate experiments. Western blot analyses of hUHRF1 and G9a expression by anti-GFP antibody are shown for each cotransfection group. ( C ) Loss of interaction between UHRF1 and G9a abolishes the UHRF1/G9a-mediated repression of p21 promoter. Reporter assays were performed as described in (B), using the wild-type G9a plasmid (EGFP-G9a) and its deletion mutant lacking the N-terminal UHRF1-interacting region (EGFP-NΔG9a). Expression of hUHRF1 and G9a/NΔG9a is shown by western blot analyses with anti-GFP antibody.

    Journal: Nucleic Acids Research

    Article Title: UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells

    doi: 10.1093/nar/gkn961

    Figure Lengend Snippet: hUHRF1 cooperates with G9a to enhance the transcriptional repression of p21 promoter. ( A ) hUHRF1-mediated transcriptional repression of the p21 promoter-luciferase reporter. COS-7 cells were cotransfected with the reporter (2 μg) and increasing amounts of EGFP-hUHRF1 as indicated at the bottom of the panel. ( B ) Enhanced transcriptional repression by G9a in the presence of exogenous hUHRF1. Luciferase activities were measured from COS-7 cells cotransfected with the same reporter described earlier and increasing amounts of EGFP-G9a (0.1–1 μg) with or without a constant amount (0.4 μg) of EGFP-hUHRF1. The luciferase assays were performed as described in Figure 4, and the data represent the means ± SD of duplicate determinations from three separate experiments. Western blot analyses of hUHRF1 and G9a expression by anti-GFP antibody are shown for each cotransfection group. ( C ) Loss of interaction between UHRF1 and G9a abolishes the UHRF1/G9a-mediated repression of p21 promoter. Reporter assays were performed as described in (B), using the wild-type G9a plasmid (EGFP-G9a) and its deletion mutant lacking the N-terminal UHRF1-interacting region (EGFP-NΔG9a). Expression of hUHRF1 and G9a/NΔG9a is shown by western blot analyses with anti-GFP antibody.

    Article Snippet: Antibodies (Ab) used for immunoprecipitation and western analyses were as follows: anti-GFP Ab (Roche Applied Science), anti-hUHRF1 Ab (BD Biosciences), anti-G9a Ab (Sigma), anti-human p21 Ab (Cell Signaling Technology), anti-mouse p21 Ab (Abcam), anti-dimethyl histone H3 (Lys9) Ab (Millipore), anti-histone H3 Ab (Cell Signaling Technology), anti-phospho-histone H3 (Ser10) Ab (Cell Signaling Technology), anti-actin Ab (Sigma) and anti-DNMT1 Ab (New England Biolabs).

    Techniques: Luciferase, Western Blot, Expressing, Cotransfection, Plasmid Preparation, Mutagenesis