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: 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