pegfp c1 er beta  (Addgene inc)

Journal: International Journal of Biological Sciences

Article Title: Estrogen Receptor Beta (ERβ) Mediated-CyclinD1 Degradation via Autophagy Plays an Anti-Proliferation Role in Colon Cells

doi: 10.7150/ijbs.30930

Figure Lengend Snippet: ERβ plays an anti-proliferation role through impairing cell cycle but not apoptosis in HCT116 cells. HCT116 cells were transiently transfected with EGFP-C1 and EGFP-C1-ERβ plasmids for 48 h, and overexpression efficiency was determined by western blotting (A) and qRT-PCR (B) . (C) Immunofluorescence analysis of HCT116 cells with Ki67 (red) and GFP (green), which were visualized by the confocal microscope. Bar graph (right) indicates the percentage of ki67 positive cells. Scale bar, 100μm. (D) Immunoblot analysis of pro-caspase-3 and cleaved-caspase-3. β-actin was used as an equal loading control. Bar graph (below) shows the relative ratio of pro-caspase-3 to cleaved-caspase-3 in HCT116. (E) Apoptosis was determined by flow cytometry after stained with Annexin V-FITC and PI. Bar graph (below) indicates the survival rate of treated cells. (F) Cell cycle distribution was analyzed by flow cytometer after DAPI stained. Bar graph (below) indicates the cell cycle phase distribution in treated cells. Immunofluorescence intensity and western blot were quantified using ImageJ software. Data shown are mean ± S.D. of three independent experiments. (*, P<0.05; **, P <0.01; ***, P<0.001).

Article Snippet: pEGFP-C1-ERβ was a gift from Michael Mancini (Addgene plasmid #28237); RFP-p62 was generously provided by professor Wei Ding (Capital Medical University, Beijing, China); RFP-LC3 and Phage-puro-ERβ fusion plasmids were constructed by our lab previously.

Techniques: Transfection, Over Expression, Western Blot, Quantitative RT-PCR, Immunofluorescence, Microscopy, Flow Cytometry, Staining, Software

Journal: International Journal of Biological Sciences

Article Title: Estrogen Receptor Beta (ERβ) Mediated-CyclinD1 Degradation via Autophagy Plays an Anti-Proliferation Role in Colon Cells

doi: 10.7150/ijbs.30930

Figure Lengend Snippet: ERβ-mediated autophagy exerts an anti-proliferation role through promoting CyclinD1 degradation in HCT116 cells. (A) Compared with Phage-puro plasmid group, the fluorescence intensity of RFP-p62 was decreased dramatically in Phage-puro-ERβ transfected group using a confocal microscope. Scale bar, 25 µm. (B) Cells were treated as described in (A), and then images were captured by the confocal microscope followed by Lyso-Tracker Red probe to stain lysosome. Scale bar, 25 µm. (C) After transfection of EGFP-C1 and EGFP-C1-ERβ plasmids for 36 h, cells were treated with CQ (10 μM) for 12 h, and then examined the expression of related-proteins. Bar graph (right) indicates the relative ratio of LAMP2, p62 and CyclinD1 and LC3-II to β-actin in HCT116. (D) CyclinD1 and ATG7 protein levels were tested in MEF Atg7 +/+ and MEF Atg7 -/- cells. Bar graph (right) indicates the relative ratio of CyclinD1 to β-actin in HCT116. (E) After ERβ transfection for 36 h, cells were treated with CQ (10 μM, 12 h), MG132 (10 μM; 12 h), or MG132 plus CQ for 12 h. Bar graph indicates the relative ratio of CyclinD1 to β-actin in HCT116. Western blot was quantified using ImageJ software. Data shown are mean ± S.D. of three independent experiments. (*, P<0.05; **, P<0.01).

Article Snippet: pEGFP-C1-ERβ was a gift from Michael Mancini (Addgene plasmid #28237); RFP-p62 was generously provided by professor Wei Ding (Capital Medical University, Beijing, China); RFP-LC3 and Phage-puro-ERβ fusion plasmids were constructed by our lab previously.

Techniques: Plasmid Preparation, Fluorescence, Transfection, Microscopy, Staining, Expressing, Western Blot, Software

Journal: International Journal of Biological Sciences

Article Title: Estrogen Receptor Beta (ERβ) Mediated-CyclinD1 Degradation via Autophagy Plays an Anti-Proliferation Role in Colon Cells

doi: 10.7150/ijbs.30930

Figure Lengend Snippet: ERβ promotes autophagy through down-regulating mTOR or BNIP3 protein in HCT116 cells. (A) Cells were treated as described in (Fig. ), then mTOR and p-mTOR were tested by immunoblotting. Bar graph (right) shows the relative ratio of mTOR and p-mTOR to β-actin in HCT116. (B) HCT116 cells were transiently transfected with EGFP-C1 and EGFP-C1-ERβ plasmids, then treated with rapamycin or not. Tagged proteins were analyzed by immunoblotting. Bar graph (right) indicates the relative ratio of LC3-II to β-actin of triplicate experiments in HCT116. (C) HCT116 cells were transiently transfected with siRNA to mTOR in HCT116 for 48 h, scrambled siRNA of mTOR as a control. mTOR and CyclinD1 antibodies were used to detect their expression levels by western blot. Bar graph (right) indicates the relative ratio of mTOR and CyclinD1 to β-actin of triplicate experiments in HCT116. (D) Tagged proteins were analyzed by immunoblotting in HCT116 treated with different concentrations of rapamycin. Bar graph indicates the relative ratio of CyclinD1 to β-actin in HCT116. (E) Cells were treated as described in (Fig. ), Then BNIP3 was tested by immunoblotting. Bar graph (right) indicates the relative ratio of BNIP3 to β-actin of triplicate experiments in HCT116. (H) Cells were transfected with Phage-puro and Phage-puro-ERβ plasmids, then the fluorescence intensity was detected by a confocal microscope. Scale bar, 25 μm. Data shown are mean ± S.D. of three independent experiments. (*, P<0.05; **, P < 0.01).

Article Snippet: pEGFP-C1-ERβ was a gift from Michael Mancini (Addgene plasmid #28237); RFP-p62 was generously provided by professor Wei Ding (Capital Medical University, Beijing, China); RFP-LC3 and Phage-puro-ERβ fusion plasmids were constructed by our lab previously.

Techniques: Western Blot, Transfection, Expressing, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: ERβ promotes Aβ degradation via the modulation of autophagy

doi: 10.1038/s41419-019-1786-8

Figure Lengend Snippet: a EGFP-C1 and EGFP-C1-ERβ groups were treated with chloroquine (10 µM) for 12 h or not. The levels of ERβ, SQSTM1, and LC3-II were compared with western blotting using related antibodies. β-actin was used as an equal loading control in this text. Densitometry analysis of LC3-II and SQSTM1 levels relative to β-actin is also shown (right). b After EBSS treatment for 1 h, cells were transfected with scrambled siRNA or ERβ siRNA for 48 h, and then cell lysates were analyzed by related antibodies. Bar graph (right) indicates the relative ratio of LC3-II, SQSTM1, and ERβ to β-actin in SH-SY5Y. c Cells were transfected with pHAGE-puro and pHAGE-puro-ERβ plasmids, the cytoplasm distribution of endogenous LC3 punctate structures was observed by confocal microscopy in SH-SY5Y cells. DNA was stained by DAPI. Scale bar = 10 μm. d Cells were transfected with EGFP-C1 and EGFP-C1-ERβ plasmids, representative electron micrographs were used to monitor autophagic vacuoles. Scale bar = 1 μm. e Cells were treated with DPN (10 nM), CQ (10 μM), and DPN plus CQ for 12 h. Cell lysates were analyzed by immunoblotting for LC3-II, SQSTM1, and β-actin protein expression. Bar graph (right) indicates the relative ratio of LC3-II and SQSTM1 to β-actin in SH-SY5Y. f Cells were treated with DPN (10 nM), the cytoplasm distribution of endogenous LC3 punctate structures was observed by confocal microscopy in SH-SY5Y cells. Scale bar = 5 μm. Data shown are mean ± S.D. of three independent experiments. (* P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: EGFP-C1-ERβ was a gift from Michael Mancini (Addgene plasmid #28237); EGFP-C1-ATG7 was kindly provided by Rongjia Zhou (Wuhan University, Wuhan, China).

Techniques: Western Blot, Transfection, Confocal Microscopy, Staining, Expressing

Journal: Cell Death & Disease

Article Title: ERβ promotes Aβ degradation via the modulation of autophagy

doi: 10.1038/s41419-019-1786-8

Figure Lengend Snippet: a Cells were treated as described in (Fig. ), protein contents of ATG7, ERβ, and ATG12–ATG5 conjugation were examined by immunoblot in SH-SY5Y cells. Bar graph indicates the relative ratio of ATG7, ATG5-ATG12 to β-actin in SH-SY5Y cells (right). b qRT-PCR was performed to measure the transactivity of ERβ, ATG7, and ATG5 in SH-SY5Y cells followed by transient transfection of EGFP-C1 and EGFP-C1-ERβ plasmids for 24 h. c Cells were treated as described in (Fig. ), immunoblot analysis was performed for ATG7, ERβ, and ATG5-ATG12 complex (left). Right panel, quantification of ATG7, ERβ, and ATG5-ATG12 complex expression using ImageJ software. d mRNA levels of ERβ, ATG7, and ATG5 in SH-SY5Y cells treated with scrambled siRNA or ERβ siRNA for 24 h. e SH-SY5Y cells were transiently transfected with pHAGE-puro or pHAGE-puro-ERβ plasmid. After 12 h transfection, cells were retransfected with ATG7 siRNA and a nonspecific RNAi control for 36 h. ATG7, ERβ, and LC3-II levels were tested by western blot (left). Right panel, quantification of LC3-II expression using ImageJ software. f pHAGE-puro or pHAGE-puro-ERβ plasmid was transfected into MEF Atg7 +/+ and MEF Atg7 − / − cells. After 48 h culture, cells were harvested for LC3 detection. Data shown are mean ± S.D. of three independent experiments. (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: EGFP-C1-ERβ was a gift from Michael Mancini (Addgene plasmid #28237); EGFP-C1-ATG7 was kindly provided by Rongjia Zhou (Wuhan University, Wuhan, China).

Techniques: Conjugation Assay, Western Blot, Quantitative RT-PCR, Transfection, Expressing, Software, Plasmid Preparation