shrna sequences targeting ezh2  (Addgene inc)

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 2. RACGAP1 overexpression promoted neuroendocrine transformation in prostate cancer. (A) Western blot analysis showed that RACGAP1 was upregulated in NEPC-like cells. (B) Immunohistochemical study of different types of prostate cancer tissues showed that RACGAP1 was highly expressed in NEPC. (C) Data for quantified immunohistochemistry in adjacent normal tissues (n=10), tumor tissues (n=10) and NEPC (n=7) of prostate cancer are shown as mean + SD. (D) Enzalutamide (MDV3100) induced the production of RACGAP1, and DHT partially reversed this effect. (E) Western blot analysis showed the protein expression of RACGAP1, CHGA, and SYP in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days. (F) The mRNA level of RACGAP1, NCAM, CHGA, SYP, and NSE in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days were determined by qRT-PCR analysis. (G) RACGAP1 and NE markers (CHGA, NCAM, NSE, and SYP) in C4-2 cells following transient transfection with control (shNC) or RACGAP1 shRNA (sh1, sh2), as detected by qRT-PCR. (H) RACGAP1 and NE markers (CHGA, SYP) in C4- 2B cells following transient transfection with RACGAP1 or an empty vector, as detected by qRT-PCR. (I) Protein

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Over Expression, Transformation Assay, Western Blot, Immunohistochemical staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Transfection, Control, shRNA, Plasmid Preparation

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 5. RACGAP1 protein interacts with the EZH2 protein and increases the protein stability of EZH2. (A) Analysis of protein-protein interaction (PPI) information shows that RACGAP1 may directly interact with EZH2. (B) GSEA shows that high RACGAP1 expression was enriched in the EZH2-TARGET pathway. (C) RNA levels of RACGAP1 and EZH2 in NE-like cells treated with RACGAP1 or empty vector (CON), as determined by qRT-PCR. (D) Western blot was performed to show protein expression of RACGAP1 and E2F1 in NE-like cells following control, RACGAP1, RACGAP1 shRNA1 (sh1), or RACGAP1 shRNA1 (sh2) transfection. (E) Total cell lysates of NE-like cells were immunoprecipitated with anti-EZH2 or anti- RACGAP1 antibodies and blotted with corresponding antibodies. (F) Representative immunofluorescence images of RACGAP1 and EZH2 protein localization in PC3 cells. (G) C4-2B-N cells and PC3 cells were transiently transfected with CON or RACGAP1 and supplied with 10 mmol/L cycloheximide (CHX), and then, total cell lysates were collected at 0, 8, 16, and 24 h after treatment. Western blot analysis was used to measure protein levels. (H) Protein expression analysis was used to calculate the half-life of EZH2 protein for C4-2B-N and PC3 cells. (I) Cells transiently transfected with RACGAP1 knockdown were treated with vehicle (DMSO), chloroquine (50 μm), or MG132 (20 μm) for 12 h. Western blotting was used to detect the protein level of RACGAP1. Bar graphs show the statistical analysis of three independent experiments. ***, p < 0.001; **, p < 0.01; *, p < 0.05, p = ns (no significance); t test for two groups or ANOVA for more than two groups.

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Control, Transfection, Immunoprecipitation, Immunofluorescence, Knockdown

Journal: Aging and disease

Article Title: Overexpression of RACGAP1 by E2F1 Promotes Neuroendocrine Differentiation of Prostate Cancer by Stabilizing EZH2 Expression

doi: 10.14336/ad.2023.0202

Figure Lengend Snippet: Figure 6. RACGAP1 stabilizes EZH2 protein expression in the ubiquitin-proteasome pathway and affects NED in prostate cancer by regulating EZH2. (A) PC3 and DU145 cells were treated with FLAG-RACGAP1 for 48 h. Total cell lysates were subjected to immunoprecipitation with EZH2 antibody and blotted with an anti- ubiquitin antibody. (B) 293T cells were co-transfected with GFP-EZH2, Myc-ubi, and different doses of FLAG- RACGAP1 (0, 2, 4 µg) for 48 h. Total cell lysates were subjected to immunoprecipitation with a GFP antibody and blotted with an anti-Myc antibody. (C) PC3 cells were transfected for rescue experiments, using PC3 cells with an shRACGAP1 plasmid control (shNC) and EZH2 expression plasmid control vector (V), cells with the shRACGAP1 plasmid (sh1) and EZH2 expression plasmid control vector (V), cells with the shRACGAP1 plasmid control (shNC) and EZH2 expression plasmid, and cells with the shRACGAP1 plasmid (sh1) and EZH2 expression plasmid. Western blotting was used to detect proteins using the indicated antibodies. We transfected PC3 and DU145 cell lines for rescue experiments, using cells with the RACGAP1 expression lentivirus control vector (V) and shEZH2 control (shNC), cells with the RACGAP1 expression lentivirus (RA) and shEZH2 control (shNC), cells with the RACGAP1 expression lentivirus control vector (V) and shEZH2, and cells with the RACGAP1 expression lentivirus (RA) and shEZH2. (D) Western blotting was used to detect proteins using the indicated antibodies. (E) Transwell assay for indicated PC3 cells (magnification: 100×). Bar graphs showing the statistical analysis of three independent experiments. ***, p < 0.001, **, p < 0.01, *, p < 0.05, p = ns (no significance); t test for two groups or ANOVA for more than two groups.

Article Snippet: The shRNA sequences targeting EZH2 were annealed and cloned into the AgeI and EcoRI sites of the plko.l plasmid (Addgene).

Techniques: Expressing, Ubiquitin Proteomics, Immunoprecipitation, Transfection, Plasmid Preparation, Control, Western Blot, Transwell Assay