Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 1. Effects of BRCA1 and HIF-1 on hypoxia-induced VEGF promoter activity. A, effect of exogenous BRCA1 and HIF-1 on VEGF reporter activity. Exponentially pro- liferating cells (MCF-7) were transfected with various combinations of pVEGF-kpnI-Luc (125 ng), HIF-1 (125 ng), and either of two doses of a BRCA1 expression vector (125 ng or 250 ng), incubated for 24 h and then exposed to hypoxia (0.1%, O2) for 6 h, harvested, and assayed for luciferase activity as described under “Experimental Procedures.” Trans- fection of HIF-1 alone into MCF-7 cells induced statistically significant pVEGF-kpnI-Luc activity under hypoxic condition (p 0.005 for comparisons of cells transfected with or without the HIF-1 expression vector in 0.1% O2) and co-transfections with both the BRCA1 and HIF-1 expression vectors further enhanced this expression (p 0.005 for comparisons between cells transfected with and without the BRCA1 expression vector, in the presence of exogenous HIF-1). B, role of the HRE in BRCA1-enhanced, HIF-1- induced VEGF promoter activity during hypoxia. To determine if the ability of BRCA1 to affectVEGFpromoteractivityduringhypoxiamightrequireinteractionbetweentheHRE and HIF-1, cells were transfected with either p11wt-Luc, p11mt-Luc, or empty vector (125 ng) plus HIF-1 and BRCA1 expression vectors (125 ng of each) as indicated in the figure, incubated for 24 h, and then exposed to hypoxia (0.1%, O2) for 6 h before meas- uring luciferase activities. As in A, BRCA1 significantly enhanced HIF-1-induced Luc activity under hypoxic conditions when the VEGF promoter contained a wild-type HRE (p 0.005 for comparisons of cells transfected with and without the BRCA1 expression vector). In contrast, neither HIF-1 alone nor HIF-1 plus BRCA1 induced statistically significant increased amounts of reporter activity in cells transfected with the HRE mutant reporter plasmid (p11mt-Luc).

Article Snippet: Transient Transfection of DNA and Luciferase Assays—The HIF-1 expression vector (pCEP4-HIF-1 ), the VEGF promoter-reporter plasmid (pVEGF-kpnI-Luc containing the 2.6-kbVEGFpromoter in pGL2), p11wt (the VEGF promoter containing the wild-type HRE in the pGL2 vector), and p11mt (the VEGF promoter containing a mutated HRE in the pGL2 vector) were purchased from ATCC.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Luciferase, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 2. Effects of BRCA1 on the endogenous levels of hypoxia-induced VEGF121 and VEGF165 mRNA. A, agarose gel analysis. Exponentially proliferating HEK293T cells were transiently transfected with empty vector (pCDNA3) or the BRCA1 expression vec- tor,incubatedfor24handthenexposedtohypoxia(0.1%,O2)for6hwhentotalRNAwas extracted used for semiquantitative RT-PCR. The gel image presented is representative of three independent experiments. B, quantitative analysis of agarose gel densitometry data. Each PCR band on photographs of the three agarose gels was quantitated using densitometry, and means S.E. of these values were calculated. BRCA1 overexpression significantly enhanced hypoxia-induced VEGF121 and VEGF165 mRNA expression (p 0.005 for comparisons of cells transfected with or without BRCA1 in 0.1% O2). C, confir- mation of PCR results. Real time PCR were performed in quadruplicate in three inde- pendent experiments to confirm the findings shown in A and B. -Actin was used as the loading control for semiquantitative RT-PCR and to normalize the quantitative real time PCR results. The results were normalized to the control (reporter only) in 21% O2 and are presented as bar graphs showing the means S.E. The effect of overexpressing BRCA1 on hypoxia-induced VEGF expression is statistically significant for both VEGF mRNAs (p 0.005).

Article Snippet: Transient Transfection of DNA and Luciferase Assays—The HIF-1 expression vector (pCEP4-HIF-1 ), the VEGF promoter-reporter plasmid (pVEGF-kpnI-Luc containing the 2.6-kbVEGFpromoter in pGL2), p11wt (the VEGF promoter containing the wild-type HRE in the pGL2 vector), and p11mt (the VEGF promoter containing a mutated HRE in the pGL2 vector) were purchased from ATCC.

Techniques: Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Real-time Polymerase Chain Reaction, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 3. The effect of hypoxia on ChIP assays for genomic VEGF promoter DNA. A, agarose gel analysis of PCR products. Exponentially proliferating cells exposed to hypoxia for the times indicated were harvested for ChIP assays as described under “Experimental Procedures.” (Protein-DNA cross-linked samples were immunoprecipi- tated with the indicated antibodies or control IgG, processed, and used as templates for PCR reactions to measure relative amounts of the HRE element-containing region of the genomicVEGFpromoterDNAthathadbeenimmunoprecipitated.)B,densitometertrac- ings of the results in A. The amount of each PCR product band was quantitated by densitometry for three independent experiments, and means S.E. were calculated. Statistically significant increases (p 0.005) of VEGF promoter DNA were observed for chromatinimmunoprecipitatedwiththeBRCA1,ARNT,andHIF-1antibodiesinhypoxic versus normoxic conditions. C, negative controls: as in A except that the primers were for genomic VEGF promoter sequences lacking known HRE sites.

Article Snippet: Transient Transfection of DNA and Luciferase Assays—The HIF-1 expression vector (pCEP4-HIF-1 ), the VEGF promoter-reporter plasmid (pVEGF-kpnI-Luc containing the 2.6-kbVEGFpromoter in pGL2), p11wt (the VEGF promoter containing the wild-type HRE in the pGL2 vector), and p11mt (the VEGF promoter containing a mutated HRE in the pGL2 vector) were purchased from ATCC.

Techniques: Agarose Gel Electrophoresis, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 4. Effect of BRCA1 mutants on hypoxia- stimulated VEGF promoter activity. A, genetic maps. Diagrams of wtBRCA1 and mutant BRCA1 (5382insC, C5365G, 5677insA, and T300G) expres- sion vector constructs. B, effect of BRCA mutants on reporter activity. MCF-7 cells were co-trans- fected with pVEGF-kpnI-Luc, HIF-1, and either wtBRCA1 or one the mutant BRCA1s as indicated. 24 h later, cells were treated with or without 0.1% hypoxic gas for 6 h. pCMV--gal and pCDNA3 vec- tors were included as controls for data normaliza- tion. The data are presented as bar graphs of the means S.E. of quadruplicate wells of three inde- pendent experiments. C, Western blot of the rela- tive wild-type and mutant BRCA1 protein levels in the cells analyzed in B.

Article Snippet: Transient Transfection of DNA and Luciferase Assays—The HIF-1 expression vector (pCEP4-HIF-1 ), the VEGF promoter-reporter plasmid (pVEGF-kpnI-Luc containing the 2.6-kbVEGFpromoter in pGL2), p11wt (the VEGF promoter containing the wild-type HRE in the pGL2 vector), and p11mt (the VEGF promoter containing a mutated HRE in the pGL2 vector) were purchased from ATCC.

Techniques: Activity Assay, Mutagenesis, Plasmid Preparation, Construct, Western Blot

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Plays a Role in the Hypoxic Response by Regulating HIF-1α Stability and by Modulating Vascular Endothelial Growth Factor Expression

doi: 10.1074/jbc.m513033200

Figure Lengend Snippet: FIGURE 6. The effect of endogenous BRCA1 levels on hypoxia-induced VEGF pro- moter activity and VEGF secretion. A, effect of BRCA1 siRNAs on VEGF promoter activ- ity. HEK293T cells were transiently transfected with control-siRNA, BRCA1-siRNA-1 or BRCA1-siRNA-3 for 48 h when the remaining siRNA was removed. The cells were then immediately co-transfected with the pVEGF-kpnI-Luc reporter construct and the same siRNAs,(freshlyprepared),incubatedfor24h,andthenexposedtohypoxiafor6hbefore luciferase activity was measured. The activity levels, relative to the control luciferase activity (control-siRNA in normoxia), are presented as bar graphs of the means S.E. of four wells from each of three independent experiments. The effects of both BRCA1 siR- NAs on hypoxia-induced VEGF-Luc reporter activity was statistically significant (p 0.005). B, effect of BRCA1 siRNAs on hypoxia-induced secretion of endogenous VEGF. VEGF secretion into the medium was measured with a VEGF ELISA kit (see “Experimental Procedures”). Cells (T47D) transiently transfected with control-siRNA, BRCA1-siRNA-1, or BRCA1-siRNA-3 for 48 h were exposed to hypoxia or normoxia for an additional 16 h (without removing the siRNAs) when the amount of VEGF secreted into the medium was measured. The values presented as bar graphs give the means S.E. of quadruplicate wells from three independent experiments. The effects of both BRCA1 siRNAs on the secretion of endogenous VEGF protein are statistically significant under hypoxic condi- tions (p 0.005) but not under normoxic conditions.

Article Snippet: Transient Transfection of DNA and Luciferase Assays—The HIF-1 expression vector (pCEP4-HIF-1 ), the VEGF promoter-reporter plasmid (pVEGF-kpnI-Luc containing the 2.6-kbVEGFpromoter in pGL2), p11wt (the VEGF promoter containing the wild-type HRE in the pGL2 vector), and p11mt (the VEGF promoter containing a mutated HRE in the pGL2 vector) were purchased from ATCC.

Techniques: Activity Assay, Transfection, Control, Construct, Luciferase, Enzyme-linked Immunosorbent 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: 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: International Journal of Molecular Sciences

Article Title: Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis

doi: 10.3390/ijms22095046

Figure Lengend Snippet: E2 induces transactivation of ERs in hGCs. Cells were transiently transfected with Firefly luciferase reporter plasmid containing or not (pGL2) estrogen-response elements (EREs) upstream of luciferase (ERE-Luc). An internal control vector containing Renilla luciferase was also included. Twenty-four hours post-transfection, cells were treated with either solvent vehicle (V) or 10 nM E2, in the presence or absence of 1 µM Fulvestrant (F), an ER degrader (SERD). Twenty-four hours later, cells were lysed and analyzed for luciferase activity. Firefly luciferase activity was normalized relative to Renilla luciferase activity, reported as relative transactivation activities arbitrarily set at 1 for the solvent vehicle (V) condition in ERE-Luc-transfected cells. Each point represents the mean ± SEM of 14 experiments (from each patient) performed in six replicates. *** p ≤ 0.001 vs. solvent vehicle (V) by two sides Wilcoxon signed-ranks test. A.U., Arbitrary Units.

Article Snippet: An empty pGL2-Firefly luciferase vector (pGL2), a pGL2 vector containing three copies of vitellogenin estrogen-response element sequence (ERE-Luc) upstream of luciferase (Addgene, Watertown, MA 02472, USA), and a pRL-SV40-Renilla luciferase (pRL-Renilla) vector (Promega, Charbonnières les bains, France) were used in the reporter assays.

Techniques: Transfection, Luciferase, Plasmid Preparation, Control, Solvent, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis

doi: 10.3390/ijms22095046

Figure Lengend Snippet: Absence of influence of ERβ2/β4/β5 on E2-induced ERβ1 transactivation in HGrC1-transfected cells. ( A ) The presence of ERα and ERβ (1, 2, 4, and 5) isoforms mRNA in hGCs ( n = 49) and HGrC1 cells ( n = 6) was evaluated by RT-qPCR (expression normalized to GAPDH). Values are represented as means ± SEM from three identical wells per patient, measured in triplicate. * p ≤ 0.05; ** p ≤ 0.01 by the Mann–Whitney test. A.U., Arbitrary Units. HGrC1 cells express low levels of endogenous ERα and ERβ isoforms, when compared to those expressed in hGCs. ( B ) HGrC1 cells were transiently cotransfected with the control Flag vector (C) alone or with the Flag-ERβ1 vector (β1), together with the Firefly luciferase reporter plasmid containing or not (pGL2) estrogen-response elements (EREs) upstream of luciferase (ERE-Luc). Flag-ERβ1 was also cotransfected (1:1 ratio) with either Flag-ERβ2 (β1+β2), Flag-ERβ4 (β1+β4), or Flag-ERβ5 (β1+β5). Renilla luciferase reporter plasmid was included as a normalizing transfection control. Twenty-four hours after solvent vehicle (V) or 10 nM E2 treatment, cells were lysed and analyzed for luciferase activity. Firefly luciferase activity was normalized to that of Renilla luciferase and reported as relative transactivation activities arbitrarily set at 1 for the control (C) solvent vehicle (V) condition in ERE-Luc-transfected cells. Each point represents the mean ± SEM in six replicates performed four times. * p ≤ 0.05; ** p ≤ 0.001 vs. vehicle (V) for each group of cotransfection, and ## p ≤ 0.001 vs. control (C) vehicle (V) using the Mann–Whitney test. A.U., Arbitrary Units. ( C ) HGrC1 cells were transiently transfected with either Flag-ERβ1 (β1), Flag-ERβ2 (β2), Flag-ERβ4 (β4), or Flag-ERβ5 (β5). Twenty-four hours post-transfection, cells were fixed, permeabilized, and the localization of Flag-ERβ proteins was monitored by immunofluorescence using an anti-Flag antibody (green). Nuclei were stained with DAPI (blue). Representative images at 20x magnification are presented.

Article Snippet: An empty pGL2-Firefly luciferase vector (pGL2), a pGL2 vector containing three copies of vitellogenin estrogen-response element sequence (ERE-Luc) upstream of luciferase (Addgene, Watertown, MA 02472, USA), and a pRL-SV40-Renilla luciferase (pRL-Renilla) vector (Promega, Charbonnières les bains, France) were used in the reporter assays.

Techniques: Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Control, Plasmid Preparation, Luciferase, Solvent, Activity Assay, Cotransfection, Immunofluorescence, Staining

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 1. Validation of the direct interaction between SANS and Magi2. (A) Domain structure schemes: SANS is composed of ankyrin repeats, CENT, SAM and a C-terminalPBM(asterisk).Magi2ischaracterizedbyGuK(guanylatekinase),PDZ1-6domainsandtwoWW(tryptophan)repeats.P:predictedphosphorylationsites (SANSS422,MagiS1152).(B)Y2H:linesin(A)indicatebaitandidentifiedprey.(C–E)GST-pulldowns:(C)FLAG-taggedSANSfull-lengthwith(3×FLAG-SANS) and without PBM (3×FLAG-SANSDPBM) were assayed with immobilized GST-PDZ6 or GST alone. Western blots revealed recovery of SANS irrespective of the PBM. (D) SANS’ N-terminus (3×FLAG-Nterm), central domain (3×FLAG-CENT), and C-terminus with/without PBM (3×FLAG-SAM-PBM/3×FLAG-SAM) were assayed. Recovery was restricted to SANS C-terminus irrespective of PBM. (E) Recovery of FLAG-tagged SANS full-length assayed with GST-PDZ4 and 5 of Magi2 failed. (F, G) Trapw assays: (F) 3×FLAG-SANS was assayed with immobilized mRFP-PDZ6. SANS was co-precipitated with Magi2-PDZ6. (G) Magi2-HA wasassayedwithimmobilizedGFP-SANS.Magi2wasco-precipitatedwith SANS.(H)Membranetargetingassay:IMCD3cellsweresingly(a;b)or co-transfected(c) with eCFP-tagged SANS N-terminally fused to a membrane-anchoring signal (MyrPalm-eCFP-SANS) or/and Magi2 C-terminally HA-tagged (Magi2-HA). (a) IMCD3 cells singly transfected with MyrPalm-eCFP-SANS (green) showed SANS enriched at the plasma membrane. (b) Cells single-transfected with Magi2-HA (red) showed a perinuclear, cytoplasmic staining. (c) In co-transfected cells, Magi2 co-localized with SANS (yellow). Nuclei were stained with DAPI. Scale bars: 10 and 2.5 mm. TCL: total cell lysate.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Biomarker Discovery, Western Blot, Transfection, Membrane, Clinical Proteomics, Staining

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 2. Phosphorylation dependency of SANS-Magi2 interaction in IMCD3 cells. (A) Cells were co-transfected with MyrPalm-eCFP-SANS and Magi2-HA and incubatedeitherwithDMSOascontrolorwithDRB.(A,a)ImmunocytochemistryofDMSO-treatedIMCD3cellsco-transfectedwithMyrPalm-eCFP-SANS(green) and Magi2-HA (red) revealed the recruitment of Magi2 towards the cell membrane. In the high magnification, the co-localization of Magi2-HA with MyrPalm-eCFP-SANS at the membrane was demonstrated (yellow). (B, b) In co-transfected cells incubated with DRB, no altered localization of Magi2-HA could be detected. High-magnification image showed no co-localization of Magi2-HA with MyrPalm-eCFP-SANS at the cell membrane. Scale bars: 10 and 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Phospho-proteomics, Transfection, Membrane, Incubation

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 3. Phosphorylation dependency of SANS-Magi2 interaction in cells transfected with phospho-/dephospho-constructs. (A) IMCD3 cells were co-transfected with a phospho-mimicking (serine mutated to glutamic acid) SANS construct (MyrPalm-eCFP-SANS S422E, green) and wild-type Magi2-HA (red). (a) High mag- nification revealed recruitment of Magi2 to the plasma membrane (yellow). (B) Cells were co-transfected with the dephospho (serine mutated to alanine)-SANS con- struct MyrPalm-eCFP-SANS S422A (green) and wild-type Magi2-HA (red). (b) High magnification demonstrated that Magi2 was no longer recruited to the membrane. (C) Cells were co-transfected with wild-type SANS (MyrPalm-eCFP-SANS, green) and a dephospho-Magi2 construct (Magi2-HA S1152A, red, serine mutated to alanine). (c) High magnification demonstrates that Magi2 was still recruited towards SANS at the membrane (yellow). Scale bars: 10 and 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Phospho-proteomics, Transfection, Construct, Clinical Proteomics, Membrane

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 5. Association of Magi2 with transferrin-positive vesicles in IMCD3 cells. (A) Uptake of Alexa647 labeled transferrin (Tf647, red) by IMCD3 cells was fluor- escence microscopically monitored during a time course of 5, 15 and 30 min, counterstained with anti-Magi2 (green). Magi2 was associated with Tf647 vesicles after 5 min of endocytoticuptakeand accumulateswith Tf647within30 min in the perinuclearcytoplasm. (B) In cells treatedwiththe GTPase inhibitordynasore uptakeof Tf647 was inhibited. Tf647 remained at the plasma membrane and Magi2 was localized in the cytoplasm. Scale bars: 10 and 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Labeling, Clinical Proteomics, Membrane

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 6. Endocytosis of transferrin is facilitated by Magi2, but decreased by SANS and phosphorylation. (A) Tf647 uptake (red) was monitored in IMCD3 cells transfected with scrambled shRNA control (green, upper panel) or shRNA against Magi2 (green, lower panel). Knock down of Magi2 diminished endocytosis of Tf647. Quantification of the mean fluorescence intensity of Tf647, normalized to mock-transfected cells revealed significant ≏57% decrease of endocytosis (SD+20.23%, n ¼ 5, P-value 0.001) in cells transfected with shRNA-Magi2 compared with cells transfected with scrambled shRNA (101%, SD+23.35%, P-value 0.032). (B) Tf647 uptake was monitored in cells transfected with scrambled shRNA control or shRNA against SANS. Knock down of SANS increased endo- cytosis of Tf647. Cytochemistry of Tf647 uptake in shRNA-transfected IMCD3 cells shows SANS-dependent endocytosis. Quantification of the mean fluorescence intensity of Tf647, normalized to mock-transfected cells revealed a significant ≏62% increase of endocytosis (SD+20.11%, n ¼ 4, P-value 0.016) in cells trans- fected with shRNA-SANS compared with mock-transfected cells and a slight increase of endocytosis of ≏30% compared with cells transfected with empty shRNA vector (SD+3.26%, P-value 0.051). (C) Tf647 uptake was monitored in IMCD3 cells treated with kinase inhibitor DRB revealing phosphorylation-dependent de- crease of endocytosis. Magi2 staining(green) illustratedthe association with Tf647. Quantification of the meanfluorescence intensity of Tf647, normalized to control revealed significant 1.63-fold increase of endocytosis (SD+0.1310, n ¼ 3, P-value 0.012) in cells treated with DRB compared with cells treated with DMSO (SD+ 0.171). Scale bar: 10 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Phospho-proteomics, Transfection, shRNA, Control, Knockdown, Plasmid Preparation, Staining

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure8. Ciliogenesisis influencedbyMagi2 anddependsonendocytosis.(A)Immunocytochemicalstainingof ciliabyantibodiesagainstacetylatedtubulin(ac.tub, red) of starved IMCD3 cells transfected with empty scrambled shRNA (control, green, upper panel) or shRNA against Magi2 (green, lower panel). Knock down of Magi2 diminished number of cells with a primary cilium. (B) Analysis of the ciliary shape of starved IMCD3 cells transfected with shRNA (b) or treated with the dynamin-inhibitor dynasore (d) revealed that the few emerging primary cilia in shRNA Magi2-transfected as well as in dynasore-treated cells are shortened (0.73 mm, SD+0.04/0.76 mm, SD+0.07) compared with controls (1.28 mm, SD+0.24/1.73 mm, SD+0.40). (C) Quantification of the total number of primary cilia in three independent experiments revealed the highly significant decrease of ciliogenesis down to 18% (SD+7.09%, n ¼ 3, P-value 0.000001) in cells transfected with shRNA Magi2, normalized to control transfected cells with scrambled shRNA (100%, SD+1.47%). (D) Quantification of the total number of primary cilia in three independent experiments revealed the highly significant decrease of ciliogenesis down to 32% (SD+3.9%, n ¼ 3, P-value 0.0003) in cells treated with dynasore compared with DMSO-treated controls (100%, SD+7.27%). Scale bars: 5 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Transfection, shRNA, Control, Knockdown

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 9. In situ localization of Magi2 and SANS-Magi2 in photoreceptor cells of murine retina. (A) Schematic representation of a rod photoreceptor cell. Ax, axoneme; CC, connecting cilium (arrow head); IS, inner segment; N, nucleus; OS, outer segment; S, synapses; 2nd, secondary neurons. (B) Indirect immunofluor- escence demonstrated Magi2 (red) localization in the IS, at the outer limiting membrane (OLM, arrow) and in the synapses of outer plexiform layer (OPL) of mouse retina. (C) Double labeling of Magi2 and compartmental marker proteins. Merged image with centrin (Cen2, green) revealed overlapping staining in the region of the CC. (D) Double labeling of Magi2 (red) and SANS (green) in the apical sub-compartment of photoreceptor cells. Magi2 and SANS were localized in the IS predom- inantlyatthebaseoftheCC(arrowhead).(E)Triplelabelingof SANS,Magi2andcentrin-2(Cen2)revealedthelocalization ofSANSandMagi2at thebaseoftheCC. (F)PLAdemonstratedSANS-Magi2complexesattheciliarybaseinsitu;(G)schematicillustrationofSANS-Magi2complexlocalizationintheciliaryregionofarod photoreceptor cell (yellow, immunofluorescence signal; red, PLA signal). BB, basal body; Ce, adjacent centriole. Scale bars: (C) 10 mm; (D) 5 mm; (F and G) 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: In Situ, Membrane, Labeling, Marker, Staining

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 10. Subcellular localization of Magi2 and TfR in mouse sections through parts of mouse rod photoreceptor cells. (A) Magi2 was detected at the outer segment (OS)base, inthe connecting cilium (CC),the adjacentcentriole(Ce) andthe innersegment(IS). (B) Electronmicrograph demonstrating the association of Magi2with vesicles in the periciliary compartment of the apical IS (white arrows). (b) Higher magnification of Magi2-labelling associated with vesicles. (C) Triple labeling of Magi2, TfR and centrin-2 (Cen2) showed localization of Magi2 and TfR at the base of the CC. (D) Immunoelectron microscopy analyses of TfR localization in a longitudinal section through the periciliary region of a mouse photoreceptor cell demonstrated TfR decoration at membranes of the apical inner segment (arrows) and the membrane (white arrowheads) of the ciliary pocket indicated by black arrowheads. (E) Immunoelectron microscopy of the cross section through the CC showed accumulation of TfR labeling in the ciliary pocket. Scale bars: (A, B, D, E) 0.25 mm; (C) 1 mm.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Labeling, Immuno-Electron Microscopy, Membrane

Journal: Human molecular genetics

Article Title: Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

doi: 10.1093/hmg/ddu104

Figure Lengend Snippet: Figure 11. Model for the SANS-Magi2 complex function in the periciliary region of photoreceptor cells. (A) Magi2 molecules mediate endocytosis from the ciliary pocket.SANSis associatedwithvesicularcargotransportthroughthe cytoplasmof theinner segment (IS)towardsthe ciliarybase. (B) In the periciliaryregion, SANS is phosphorylated by protein kinase CK2 increasing its affinity to Magi2. (C) Phosphorylated SANS recruits Magi2 from the endocytosis module of the ciliary pocket and facilitates cargo vesicle targeting to the periciliary membrane and thereby exocytosis. OS, outer segment.

Article Snippet: Human SANS (amino acid 2–461) and murine Magi2 (amino acid 2–1112) were subcloned in the MyrPalm-eCFP vector (plasmid 14867, Addgene, Cambridge, MA, USA) (64) containing an N-terminal membrane-anchoring peptide and eCFP.

Techniques: Membrane