Journal: Cell death discovery

Article Title: p21 Regulates Wnt-Notch balance via DREAM/MMB/Rb-E2F1 and maintains intestinal stem cell homeostasis.

doi: 10.1038/s41420-024-02192-z

Figure Lengend Snippet: Fig. 4 The activated E2F1 up-regulated the transcription of Notch1, resulted in the activation of Notch pathway, and inhibition of Wnt pathway in p21G2TKO MEFs. A Co-IP with anti-E2F1 antibody indicate that E2F1 binding Rb reduced in p21G2TKO cells. B ChIP assay with anti-E2F1 antibody reveals that E2F1 binds Notch1 promoter with higher efficiency in p21G2TKO cells, suggesting the released E2F1 activity promoted Notch1 transcription. N = 3, **, p < 0.01; ***, p < 0.001. C Co-IP with anti-β-Catenin antibody indicated more Notch1 and Rbpj proteins are bound to β-Catenin in p21G2TKO cells, suggesting that the up-regulation of Notch1 results in its enhanced binding to β-Catenin. D ChIP assay with anti-β-Catenin antibody showed that more β-Catenin bound to Hes1 promoter in p21G2TKO cells. N = 3, *, p < 0.05; **, p < 0.01.

Article Snippet: After sonication, the supernatant was collected and incubated with ChIP grade protein A magnetic beads and anti-E2F1 antibody (Novus, used 2 μg for 20 μg of chromatin DNA), or anti-β-Catenin antibody (non-phosphorylated (active), CST, used 2 μg for 20 μg of chromatin DNA) overnight at 4 °C.

Techniques: Activation Assay, Inhibition, Co-Immunoprecipitation Assay, Binding Assay, Activity Assay

Journal: Cell death discovery

Article Title: p21 Regulates Wnt-Notch balance via DREAM/MMB/Rb-E2F1 and maintains intestinal stem cell homeostasis.

doi: 10.1038/s41420-024-02192-z

Figure Lengend Snippet: Fig. 5 Overexpression of p21 rescued the imbalance of Wnt-Notch, and shifted the DREAM/MMB/Rb-E2F1 complex towards its inhibitory function. A Overexpression of p21 in p21G2TKO cells restored the expression of Wnt3, β-Catenin proteins, and suppressed the expression of Nocth1, Hes1, and Rbpj. B Overexpression of p21 suppressed the expression of MMB/E2F1 proteins and restored the inhibition DREAM/Rb proteins. C The MMB down-stream proteins and cell cycle related proteins were also suppressed by p21 overexpression. D Immunofluorescence staining indicated the up-regulation of Wnt3 and β-Catenin, and suppression of Nocth1, Hes1 protein level after overexpression of p21.

Article Snippet: After sonication, the supernatant was collected and incubated with ChIP grade protein A magnetic beads and anti-E2F1 antibody (Novus, used 2 μg for 20 μg of chromatin DNA), or anti-β-Catenin antibody (non-phosphorylated (active), CST, used 2 μg for 20 μg of chromatin DNA) overnight at 4 °C.

Techniques: Over Expression, Expressing, Inhibition, Staining

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of cytoplasmic (Cyto.) and nuclear (Nuc.) fractions of uninfected or HSV-1(F)-infected HeLa and HEp-2 cell lysates separated on bisacrylamide gels and reacted with antibody to E2F-1 as described in Materials and Methods. The cells were infected at time zero and harvested at the indicated times after infection.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of cytoplasmic and nuclear fractions of uninfected or HSV-1(F)-infected serum-starved, contact-inhibited HEL fibroblasts lysates separated on bisacrylamide gels and reacted with antibody to E2F-1. Cells were infected at time zero and harvested at the indicated times after infection.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of nuclear fractions of uninfected or HSV-1(F)-infected HEp-2 cell lysates separated on bisacrylamide gels and reacted with antibody to E2F-2. Cells were infected at time zero and harvested at the indicated times after infection.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of nuclear fractions of uninfected or HSV-1(F)-infected serum-starved, contact-inhibited HEL fibroblasts lysates separated on bisacrylamide gels and reacted with antibody to E2F-2. Cells were infected at time zero and harvested at the indicated times after infection.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of cytoplasmic and nuclear fractions of uninfected or HSV-1(F)-infected HEp-2 cell lysates treated with or without CIAP, separated on bisacrylamide gels, and reacted with antibody to E2F-4. Cells were infected at time zero and harvested at the indicated times after infection. The boxed region indicates the isoforms of E2F-4 detected.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of cytoplasmic and nuclear fractions of uninfected or HSV-1(F)-infected serum-starved, contact-inhibited HEL fibroblasts lysates separated on bisacrylamide gels and reacted with antibody to E2F-4. Cells were infected at time zero and harvested at the indicated times after infection. Multiple E2F-4-reactive bands were observed as indicated by the vertical lines.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of nuclear fractions of uninfected or HSV-1(F)-infected HEp-2 cell lysates separated on bisacrylamide gels and reacted with antibody to E2F-5. Cells were infected at time zero and harvested at 14 h after infection.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of cytoplasmic and nuclear fractions of uninfected or HSV-1(F)-infected serum-starved, contact-inhibited HEL fibroblasts lysates separated on bisacrylamide gels and reacted with antibody to E2F-5. Cells were infected at time zero and harvested at the indicated times after infection.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: Immunoblot of nuclear fractions of uninfected or HSV-1(F)-infected HEp-2 cell lysates treated with or without CIAP, separated on bisacrylamide gels, and reacted with antibody to E2F-1. Cells were infected at time zero and harvested at the indicated times after infection.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Western Blot, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: E2F DNA electrophoretic mobility shift assay of cytoplasmic and nuclear fractions of uninfected or HSV-1-infected HeLa cell lysates. Cells were infected at time zero and harvested at the indicated times. Increasing amounts of protein extract (0.5, 1, 5, and 10 μg) were reacted with 32P-end-labeled DNA with the E2F consensus binding site. To determine if the gel shift bands were competable, 100-fold excess unlabeled probe was added to 10 mg of each extract (lanes 5, 10, 15, and 20). Bands that were competable are labeled with a dark line.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Electrophoretic Mobility Shift Assay, Infection, Labeling, Binding Assay

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: E2F DNA electrophoretic mobility shift and supershift assays of nuclear fractions of uninfected or HSV-1(F)-infected HEp-2 cell lysates. Cells were infected at time zero and harvested at indicated times; 5 μg of protein extract was subjected to gel shift assay without antibody (Ab.; lanes 1 and 2). Supershift assays were done with the indicated antibodies as described in Materials and Methods (lanes 3 to 10).

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Electrophoretic Mobility Shift Assay, Infection

Journal:

Article Title: E2F Proteins Are Posttranslationally Modified Concomitantly with a Reduction in Nuclear Binding Activity in Cells Infected with Herpes Simplex Virus 1

doi:

Figure Lengend Snippet: E2F DNA electrophoretic mobility shift assays of cytoplasmic (A) and nuclear (B) fractions of uninfected or HSV-1(F)-infected serum-starved, contact-inhibited HEL fibroblasts lysates. Cells were infected at time zero and harvested at indicated times; 5 μg of protein extract was subjected to gel shift assay. Circles are next to bands specific to either uninfected or HSV-1(F)-infected lysates. Competable gel shift bands were determined by the addition of 100-fold excess unlabeled DNA probe (lanes 5, 6, 12, and 13). Labeled probe without protein extract was loaded in lanes 7 and 14. (C) E2F DNA electrophoretic mobility supershift assays with nuclear lysate of uninfected HEL fibroblast lysates. The square shows the shift upward of a E2F-2–DNA complex.

Article Snippet: E2F consensus DNA binding oligonucleotide was purchased from Santa Cruz Biotechnology (sc-2507).

Techniques: Electrophoretic Mobility Shift Assay, Infection, Labeling

Journal: Cell Death Discovery

Article Title: p21 Regulates Wnt-Notch balance via DREAM/MMB/Rb-E2F1 and maintains intestinal stem cell homeostasis

doi: 10.1038/s41420-024-02192-z

Figure Lengend Snippet: A The comparison of morphology (H&E), cell apoptosis (cleaved caspase3, Tunel), cell proliferation (phosphorylated H3 and BrdU), and mobilization (BrdU) occurring in the intestine villi of different genotype of mice reveal the fast turnover of intestinal epithelia in p21G2TKO mice. B The quantification of ( A ). N = 3, ns, no significance; **, p < 0.01; ***, p < 0.001. C The immunohistochemistry staining reveals a robust increase of Bmi1 + cells (+4 stem cell marker) in the crypts of p21G2TKO mice, which partially overlapped with the proliferation marker Ki67 by staining in serial sections. The red frames show the similar structures across serial sections, and the red arrows point to colocalization. D The quantification of ( C ). N = 3, ns, no significance; ***, p < 0.001. E The Western blotting of isolated crypt protein extract indicate an increased expression of Bmi1 in both p21 null and p21G2TKO (inverse expression pattern with Olfm4 in Fig. ), while the phosphorylated H3 and PCNA were only up-regulated in p21G2TKO crypts. F The Western blotting of proteins from MEFs indicate the expression of proteins favors the MMB/E2F1 complex than the DREAM/Rb complex in p21G2TKO cells. G The Western blotting of proteins from MEFs indicate the down-stream proteins of DREAM/MMB/Rb-E2F1 pathway is shifted towards cell cycle promoting form (MMB/E2F1) in p21G2TKO cells. H Co-IP confirm that the binding pattern of DREAM/MMB/Rb-E2F1 complex shifted towards the active form for cell cycle progression in p21G2TKO cells. I The cell cycle analysis reveals that p21G2TKO cells contained lesser percentage of G1 phase cells and higher percentage of S phase cells.

Article Snippet: After sonication, the supernatant was collected and incubated with ChIP grade protein A magnetic beads and anti-E2F1 antibody (Novus, used 2 µg for 20 µg of chromatin DNA), or anti-β-Catenin antibody (non-phosphorylated (active), CST, used 2 µg for 20 µg of chromatin DNA) overnight at 4 °C.

Techniques: Comparison, TUNEL Assay, Immunohistochemistry, Staining, Marker, Western Blot, Isolation, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Cell Cycle Assay

Journal: Cell Death Discovery

Article Title: p21 Regulates Wnt-Notch balance via DREAM/MMB/Rb-E2F1 and maintains intestinal stem cell homeostasis

doi: 10.1038/s41420-024-02192-z

Figure Lengend Snippet: A Co-IP with anti-E2F1 antibody indicate that E2F1 binding Rb reduced in p21G2TKO cells. B ChIP assay with anti-E2F1 antibody reveals that E2F1 binds Notch1 promoter with higher efficiency in p21G2TKO cells, suggesting the released E2F1 activity promoted Notch1 transcription. N = 3, **, p < 0.01; ***, p < 0.001. C Co-IP with anti-β-Catenin antibody indicated more Notch1 and Rbpj proteins are bound to β-Catenin in p21G2TKO cells, suggesting that the up-regulation of Notch1 results in its enhanced binding to β-Catenin. D ChIP assay with anti-β-Catenin antibody showed that more β-Catenin bound to Hes1 promoter in p21G2TKO cells. N = 3, *, p < 0.05; **, p < 0.01.

Article Snippet: After sonication, the supernatant was collected and incubated with ChIP grade protein A magnetic beads and anti-E2F1 antibody (Novus, used 2 µg for 20 µg of chromatin DNA), or anti-β-Catenin antibody (non-phosphorylated (active), CST, used 2 µg for 20 µg of chromatin DNA) overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Activity Assay

Journal: Cell Death Discovery

Article Title: p21 Regulates Wnt-Notch balance via DREAM/MMB/Rb-E2F1 and maintains intestinal stem cell homeostasis

doi: 10.1038/s41420-024-02192-z

Figure Lengend Snippet: A Overexpression of p21 in p21G2TKO cells restored the expression of Wnt3, β-Catenin proteins, and suppressed the expression of Nocth1, Hes1, and Rbpj. B Overexpression of p21 suppressed the expression of MMB/E2F1 proteins and restored the inhibition DREAM/Rb proteins. C The MMB down-stream proteins and cell cycle related proteins were also suppressed by p21 overexpression. D Immunofluorescence staining indicated the up-regulation of Wnt3 and β-Catenin, and suppression of Nocth1, Hes1 protein level after overexpression of p21.

Article Snippet: After sonication, the supernatant was collected and incubated with ChIP grade protein A magnetic beads and anti-E2F1 antibody (Novus, used 2 µg for 20 µg of chromatin DNA), or anti-β-Catenin antibody (non-phosphorylated (active), CST, used 2 µg for 20 µg of chromatin DNA) overnight at 4 °C.

Techniques: Over Expression, Expressing, Inhibition, Immunofluorescence, Staining

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: Differential gene expression in response to DTX-ENZ combined treatment. ( A ) Flowchart of RNA sample preparation and RNA-seq. ( B ) Analysis of common deregulated genes in double drugs v.s. Control or ENZ or DTX group. ( p < 0.05, FDR < 0.25, fold change >1.4) ( C ) Expression of E2F1 in R1-ADR cells 12 h after drug treatment were determined by qRT-PCR. Error bars, S.D. * p < 0.05. ( D ) Protein expression of E2F1 in R1-ADR cells 12 and 24 h after drug treatment were determined by western blot. ( E ) Re-expressed E2F1 partially rescued DTX+ENZ induced growth inhibition in R1-ADR cells. Cell growth was determined by CCK-8 assays 72 h after re-introduced exogenous E2F1 in the presence of DTX and ENZ. Error bars, S.D. * p < 0.05.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Gene Expression, Sample Prep, RNA Sequencing, Control, Expressing, Quantitative RT-PCR, Western Blot, Inhibition, CCK-8 Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: AR3 and AR-FL differentially regulated E2F1 expression. ( A , B ) R1-ADR cells were infected with the lentivirus encoding the control shRNA, AR3 shRNA (shAR3) or AR-FL shRNA (shAR-FL) for 48 h. Protein levels of E2F1, AR-FL, and AR3 were determined by western blot ( A ) and mRNA levels were determined by qRT-PCR ( B ), Error bars, S.D. * p < 0.05. ( C ) Binding of AR3 or AR-FL to the putative ARE sites of human E2F1 gene regulatory region was analyzed by ChIP assays. Error bars, S.D. * p < 0.05. ( D ) The E2F1-promoter driven luciferase reporter (E2F1-LUC) activity was measured in HEK293T cells treated with 0.1nM DHT or/and 10 μM ENZ. ( E ) ARR2-promoter luciferase assay was performed in HEK293T cells treated with 0.1nM DHT or/and 10 μM ENZ.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Expressing, Infection, Control, shRNA, Western Blot, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: AR3 and AR-FL recruited different cofactors and differentially regulated E2F1 gene expression. ( A ) R1-ADR cells were infected with the lentivirus encoding the control shRNA, shAR3 or shAR-FL for 48 h. Cell lysates were then immunoprecipitated with an anti-AR or anti-E2F1 antibody followed by western blot. ( B ) HEK293T cells were cotransfected with AR-FL, AR3, and/or E2F1-HA. Cell lysates were then immunoprecipitated with an anti-AR or anti-HA antibody followed by western blot. ( C ) R1-ADR cells were infected with the lentivirus encoding the control shRNA or shAR3 or shAR-FL for 48 h and then treated with 10nM DHT for 2 h before collection. Cell lysates were then immunoprecipitated with an anti-AR or anti-Rb antibody followed by western blot. ( D ) E2F1-promoter luciferase assay was performed in HEK293T cells treated with 0.1nM DHT or/and 10 μM ENZ. ( E ) E2F1-promoter luciferase assays with mutant E2F1 were performed in HEK293T cells.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Gene Expression, Infection, Control, shRNA, Immunoprecipitation, Western Blot, Luciferase, Mutagenesis

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: E2F1 regulated AR expression. ( A ) AR-FL, AR3, and E2F1 protein level 4 days after DTX+ENZ treatment were determined by western blot. ( B , C ) R1-ADR cells were infected with the lentivirus encoding the control shRNA or E2F1 shRNA for 48 h. Expression level of E2F1, AR-FL, and AR3 were determined by qRT-PCR ( B ), Error bars, S.D. * p < 0.05 and western blot ( C ). ( D ) Binding of E2F1 to the regulatory regions of AR was analyzed by ChIP assay.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Expressing, Western Blot, Infection, Control, shRNA, Quantitative RT-PCR, Binding Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: E2F1 and AR/ARv expression was recovered in R1-DDR cells. ( A ) RNA-seq analysis, heatmap of those commonly dysregulated genes in R1-ADR cells treated with DTX+ENZ (p < 0.05, FDR < 0.25, fold change >1.4), we compared R1-DDR cells (maintained in DTX+ENZ) to R1-ADR cells treated with DTX+ENZ (D+E). Missing values are in color “gray”. ( B ) protein level of AR/ARv and E2F1 in R1-DDR cells were determined by western blot. ( C ) R1-DDR cells infected with lentivirus encoding control or shE2F1 or shAR-FL or shAR3. Cell growth was determined by CCK-8 assays 3 days after infection.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: Expressing, RNA Sequencing, Western Blot, Infection, Control, CCK-8 Assay

Journal: Cells

Article Title: The Role of Crosstalk between AR3 and E2F1 in Drug Resistance in Prostate Cancer Cells

doi: 10.3390/cells9051094

Figure Lengend Snippet: Auranofin inhibited the growth of double drug-resistant prostate cancer cells in vitro and in vivo. ( A ) R1-DDR (left panel) and LN-DDR (right panel) cells were treated with different doses of Auranofin for 72 h. Cell growth was determined by CCK8 assay. ( B ) R1-DDR and LN-DDR cells were treated with 2 μM Auranofin for 12 h, Protein level of E2F1, AR-FL, and AR3 in R1-DDR cells were determined by western blot. ( C ) Animals were treated with solvent or DTX (5 mg/kg once a week) + ENZ (10 mg/kg per day, 5 days per week) or auranofin (5 mg/kg per day, 5 days per week), n = 5 per group. Error bars, S.E.M. * p < 0.05 ( D ) TUNEL assay was performed to detect apoptosis of R1-DDR xenograft. Error bars, S.D. * p < 0.05. ( E ) Ki67 proliferation marker was examined by immunohistochemistry. ( F ) AR/ARv and E2F1 protein level in R1-DDR xenograft were determined by western blot.

Article Snippet: The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24225) [ ], the E2F1 DNA binding mutant vector pCMV E2F1 E132 was a gift from Kristian Helin (Addgene, Cambridge, MA, USA, plasmid # 24224) [ ], the E2F1 promoter luciferase vector pGL2-AN was a gift from William Kaelin (Addgene, Cambridge, MA, USA, plasmid # 20950) [ ], and the AR expression vector and ARR2-Luc vector were generated as previously described [ , ].

Techniques: In Vitro, In Vivo, CCK-8 Assay, Western Blot, Solvent, TUNEL Assay, Marker, Immunohistochemistry

Journal: Cell Cycle

Article Title: Periodic expression of Kv10.1 driven by pRb/E2F1 contributes to G2/M progression of cancer and non-transformed cells

doi: 10.1080/15384101.2016.1138187

Figure Lengend Snippet: Kv10.1 expression in G2/M depends on pRb/E2F1 activity. A. Schematic of human Kv10.1 promoter indicating E2F1 responsive element upstream of the transcription starting site (TSS). B. Luciferase activity driven by Kv10.1 promoter (black trace) showed a peak activity during G2/M transition (One-Way ANOVA, P < 0.0001). Mutation of E2F1 responsive element (red trace) abolished promoter activity (Two-way ANOVA. Changes in expression levels with time after release P < 0.0001, mutated vs. wild type Kv10.1 promoter P < 0.0001). C. HPV-E7 overexpression increased Kv10.1 promoter activity (Two-way ANOVA. Changes in expression levels with time after release P < 0.01, effect of HPV-E7 overexpression P < 0.0001). D. Analysis by SDS-PAGE and Western blotting using anti-Kv10.1, anti-pRb and anti-E2F1 showed that Kv10.1 expression profile is delayed from E2F1 peak expression. E. E2F1 binding to endogenous Kv10.1 promoter (One-Way ANOVA, P < 0.0001) and Cyclin A2 promoter (One-Way ANOVA, P < 0.001) during G2/M transition (8 h). Cyclin A2, an E2F1 target gene regulated at the G2/M transition was used here as a positive control. Fold enrichment was calculated relative to GAPDH promoter signal. GAPDH is a non-E2F1 regulated gene. All experiments were performed at least 3 times.

Article Snippet: For E2F1 and HPV E7 overexpression, DNA plasmids 408 pSG5L HA E2F1 Addgene p10736 https://www.addgene.org/Plasmid10736/ , p1324 HPV-16 E7 Addgene p8643 https://www.addgene.org/Plasmid8643/ and pGL3 5′UTR- KCNH1 were transfected (1 μg DNA/200.000 cells) using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Activity Assay, Luciferase, Mutagenesis, Over Expression, SDS Page, Western Blot, Binding Assay, Positive Control