cdnas of mouse e- cadherin nm_004360.5  (Addgene inc)

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: E-cadherin expression is increased in metastases compared to primary tumors . A) Percentage of E-cadherin-positve cells is increased in metastases compared to the primary tumors. Organ sites of metastases are organized by color: liver (red), lung (blue), and brain (yellow) B) Example of a case showing strong expression of E-cadherin in the metastasis (right) compared to negative expression in primary (left). C) Heterogeneous expression of E-cadherin in the center (dashed inset) versus edge (solid inset) of a liver metastasis. "C" denotes tumor and "H" denotes hepatocytes. D) Quantification of E-cadherin staining in the center and edge of the liver metastasis.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Expressing, Staining

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: E-cadherin expression alters cell morphology . A) Cell morphology as examined by phase contrast microscopy (left column) and E-cadherin expression (red) as detected by immunofluorescence (right column) B) Immunblot analysis illustrates ectopic expression of E-cadherin in 231-Ecad cells as well as expression of various epithelial and mesenchymal markers in the E-cadherin mutants. C) Immunofluorescence of vimentin, cytokeratin-18 and actin cytoskeleton (rhodamine phalloidin). Shown are representative of at least three different assessments using one of two independent clones of each cell variant. D) Quantification of fluorescence using ImageJ, n = 20 cells, p < 0.05.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Expressing, Microscopy, Immunofluorescence, Clone Assay, Variant Assay, Fluorescence

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: E-cadherin expression suppresses migration (A) and invasion (B) . Cell migration was analyzed using a scratch assay. Scratch closure was measured over a period of 24 hours and the fraction closure was quanitified by Metamorph software (n = 3). Invasion was measured in using a Matrigel invasion assay in which cells were allowed to migrate through a Matrigel-coated transwell insert for a period of 24 hours. N = 3 in triplicate; mean ± s.e.m. Results shown are representative of one of two independent clones of each mutant.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Expressing, Migration, Wound Healing Assay, Software, Invasion Assay, Clone Assay, Mutagenesis

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: Hepatocytes drive the re-expression of E-cadherin in MDA-MB-231 breast cancer cells . A) Immunoblot of proteins lysates from MDA-MB-231/hepatocyte co-cultures using a human-specific antibody. B) Flow cytometry of the MDA-MB-231 population using a human-specific antibody shows a unimodal population on day 0 and a bimodal population on day 6. C) MDA-MB-231 cells do not express E-cadherin without hepatocytes. D) RT-PCR using human-specific primers of MDA-MB-231 cells after 6 days of co-culture with hepatocytes. Means (n = 4) ± s.d. Note that species-specific primers do not amplify E-cadherin or GAPDH from hepatocytes.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Expressing, Western Blot, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Co-Culture Assay

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: Breast cancer cells culture with hepatocytes re-express E-cadherin but maintain vimentin A) Immunostaining of RFP-labeled breast cancer cells in hepatocyte coculture; E-cadherin (green), RFP (red), DAPI (blue) B) Immunostaining for vimentin (green), RFP (red), DAPI (blue) . Shown are representative of at least three different assessments using at least two independent clones of each cell variant.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Immunostaining, Labeling, Clone Assay, Variant Assay

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: A subset of primary breast carcinoma explants re-express E-cadherin when cocultured with primary hepatocytes . A) Flow cytometry analysis of primary explants using a human-specific E-cadherin antibody. A fluorescence unit of 1 indicates that the fluorescence intensity was equal to the same gate performed without addition of antibody. B) Confocal microscopy of two positive explants. Explants (C), hepatocytes (H). Human-specific E-cadherin, blue; actin, red; nuclei, green.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Flow Cytometry, Fluorescence, Confocal Microscopy

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: Breast cancer cells lose methylation of E-cadherin promoter methylation following hepatocyte coculture . A) Nested PCR method to detect methylation status of the E-cadherin promoter in a six day time course of hepatocye coculture. Above, bisulfite-treated DNA is amplified with primers that exclude CpG islands to amplify a 270 bp region independent of methylation status. Below, nested primers anneal to the 270 bp target to amplify a methylated (112 bp) or unmethylated (120 bp) fragment in the six day time course. MCF7 is used an unmethylated control. B) Bisulfite sequencing of CpG islands in the E-cadherin promoter. Figure adapted from Corn et al . CpG islands are indicated as vertical lines on map; each CpG island is represented a circle. MCF7, MDA-MB-231, and MDA-MB-435 were sequenced on days 1,3, and 5 coculture. Open circle, unmethylated CpG; closed circle, methylated CpG; filled circle, mixed quality values.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Methylation, Nested PCR, Amplification, Methylation Sequencing

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: Re-expression of E-cadherin follows a proliferation-dependent demethylation of the E-cadherin promoter . A) MS-PCR of MDA-MB-231 cultured alone in hepatocyte growth media B) MS-PCR using human-specific primers that amplify the imprinted H19 gene. C) MS-PCR of E-cadherin promoter following addition of MMC D) Addition of MMC prevents E-cadherin re-expression at the protein level. E) The maintenance demethylase DNMT1 does not change in localization or intensity in MDA-MB-231 cancer cells when cocultured with hepatocytes. DNMT1, red; DAPI, blue.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Expressing, Cell Culture

Journal: Molecular Cancer

Article Title: Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition

doi: 10.1186/1476-4598-9-179

Figure Lengend Snippet: E-cadherin positive metastatic foci originate from E-cadherin negative primary tumors . A) Left, human MDA-MB-231 breast cancer cell xenograft in a mouse inguinal fat pad (H&E); middle, human-specific E-cadherin antibody indicates the absence of E-cadherin expression in the center of the primary tumor; right, absence of human-specific E-cadherin labeling at the periphery of the tumor. B) Micrometastases in the lung originating from the primary xenograft in A . Immunoperoxidase labeling of diseased portions of the mouse lung indicate the presence of human E-cadherin-positive MDA-MB-231 cancer cells; bottom adjacent.

Article Snippet: 231-Ecad cells were made by co-transfecting a plasmid encoding the E-cadherin full-length cDNA sequence (Open Biosystems) with the pcDNA 3.1 plasmid (Invitrogen) and cultured in 900 μg/ml G418 to select for stable transfectants.

Techniques: Expressing, Labeling

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet: Ligand-activated PPARA strongly represses CDH1 expression Ppara +/+ and Ppara −/- mice fed either control chow diet or a diet containing 0.1% Wy14,643 or 0.5% fenofibrate for 48 h. (A) Venn diagrams of RefSeq transcripts were repressed by Wy14,643 or fenofibrate (fold change < −3 at FDR <0.05) in Ppara +/+ and Ppara −/- mouse livers, as determined by RNA-seq. n = 10–15 mice per group. (B) Heatmap of tumor suppressor gene expression provided from RNA-seq analyses in Wy14,643-treated Ppara +/+ and Ppara −/- mouse livers. n = 10–15 mice per group. Color key, Log2 fold-change (FC). (C) RT-qPCR analysis of Cdh1 mRNA in Wy14,643- or fenofibrate-treated Ppara +/+ and Ppara −/- mouse livers. n = 4 mice per group. (D and E) Western blot analysis (D) and the quantification (E) of CDH1 protein in Wy14,643-treated Ppara +/+ and Ppara −/- mouse livers. n = 3 mice per group. (F) Heatmap of gene expression in the CDH1-related gene and PPARA target genes provided from RNA-seq analyses of Wy14,643-treated Ppara +/+ and Ppara −/- mouse livers. n = 10–15 mice per group. Color key, Log2 fold-change (FC). Each data point represents the mean ± SEM Significant differences from normal chow diet-treated each genotype mouse livers: ∗ p < 0.001.

Article Snippet: pCMV3-mouse CDH1 , Sino Biological , Cat# MG50671-CF.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet: PPARA activation causes methylation of the Cdh1 promoter marked on H3K9me3 in a UHRF1-dependent manner (A) Model of gene repression by UHRF1. (B) Heatmap of UHRF1-related gene expression provided from RNA-seq analyses in Wy14,643-treated Ppara +/+ and Ppara −/- mouse livers. Mice fed either control chow diet or a diet containing 0.1% Wy14,643 for 48 h. n = 10–15 mice per group. Color key, Log2 fold-change (FC). (C) H3K9me3 ChIP-seq reads peaks in 5′ upstream region of Cdh1 in mouse livers. (D) Heatmap of gene expression in the H3K9me3 binding peak provided from RNA-seq analyses in Wy14,643-treated Ppara +/+ and Ppara −/- livers. Mice fed either a control chow diet or a diet containing 0.1% Wy14,643 for 48 h. n = 10–15 mice per group. (E and F) UHRF1, DNMT1, HDAC1, H3K9me3, or SETDB1 ChIP-qPCR (E) or methyl-DNA immunoprecipitation analysis (F) assessed Cdh1 promoter in liver samples from Ppara +/+ and Ppara −/- mice treated with Wy14,643. Mice fed either control chow diet or a diet containing 0.1% Wy14,643 for 48 h. n = 3–4 mice per group. Significant differences from normal chow diet-treated each genotype mouse livers: ∗ p < 0.05.

Article Snippet: pCMV3-mouse CDH1 , Sino Biological , Cat# MG50671-CF.

Techniques: Activation Assay, Methylation, Expressing, RNA Sequencing Assay, ChIP-sequencing, Binding Assay, Immunoprecipitation

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet: Repression of CDH1 expression by ligand-activated PPARA is reversed by the knockdown of E2F8 or UHRF1 Mice were fed either control chow diet or a diet containing 0.1% Wy14,643 for 48 h. (A and B) Western blot analysis (A) and the quantification (B) of E2F8, UHRF1, and CDH1 proteins in AAV-shRNA-Control (Control) or AAV-shRNA-E2f8 (shE2f8)injected mice treated with Wy14,643. n = 3 mice per group. (C) qRT-PCR analysis of E2f8 , Uhrf1 , and Cdh1 mRNA in AAV-shRNA-Control (Control) or AAV-shRNA-E2f8 (shE2f8) injected mice treated with Wy14,643. n = 4 mice per group. (D) UHRF1 ChIP-qPCR assessed Cdh1 or Rpl30 promoter region binding in liver samples from AAV-shRNA-Control (Control) or AAV-shRNA-E2f8 (shE2f8) injected mice treated with Wy14,643. n = 4 mice per group. (E and F) Western blot analysis (E) and the quantification (F) of E2F8, UHRF1, and CDH1 proteins in AAV-shRNA-Control (Control) or AAV-shRNA-Uhrf1 (shUhrf1) injected mice treated with Wy14,643. n = 3 mice per group. (G) qRT-PCR analysis of E2f8 , Uhrf1 , and Cdh1 mRNA in AAV-shRNA-Control (Control)- or AAV-shRNA-Uhrf1 (shUhrf1)injected mice treated with Wy14,643. n = 4 mice per group. (H) UHRF1 ChIP-qPCR assessed Cdh1 or Rpl30 promoter region binding in liver samples from AAV-shRNA-Control (Control) or AAV-shRNA-Uhrf1 (shUhrf1) injected mice treated with Wy14,643. n = 4 mice per group. Each data point represents the mean ± SEM Significant differences from shE2f8-or shUhrf1-treated mouse livers: ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: pCMV3-mouse CDH1 , Sino Biological , Cat# MG50671-CF.

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

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet: PPARA autoinduction accelerates the E2F8-UHRF1-CDH1 pathway (A) qRT-PCR analysis of Ppara mRNA in Wy14,643- or fenofibrate-treated Ppara +/+ and Ppara −/- mouse livers. Ppara +/+ and Ppara −/- mice fed either control chow diet or a diet containing 0.1% Wy14,643 or 0.5% fenofibrate for 48 h. n = 4 mice per group. Significant differences from normal chow diet-treated each genotype mouse livers: ∗ p < 0.001. (B and C) Western blot analysis (B) and the quantification (C) of PPARA protein in Wy14,643-treated Ppara +/+ and Ppara −/- mouse livers. Ppara +/+ and Ppara −/- mice fed either a control diet or a diet containing 0.1% Wy14,643 for 48 h. n = 3 mice per group. Significant differences from normal chow diet-treated each genotype mouse livers: ∗ p < 0.01. (D) qRT-PCR analysis of Ppara , Cyp4a10 , Acox1 , Acot1/2 , E2f8 , Uhrf1, and Cdh1 mRNAs in livers of Ppara +/+ mice treated by gavage with Wy14,643 (50 mg/kg). Livers were collected at t = 0, 1.5, 3, 6, 12, and 24 h. n = 4 mice per group. Significant differences from Wy14,643-treated mouse livers (0 h): ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (E) PPARA ChIP-seq reads peaks in the Ppara and Acox1 genes from Wy14,643-treated mouse livers. Mice were treated with Wy14,643 (50 mg/12 h/kg for 24 h) or vehicle by oral gavage and administered either a normal diet or a 0.1% Wy14,643-chow diet for 24 h. n = 3 mice per group. (F) Schematic representation of the positions of the putative five PPRE sequences contained in the Ppara gene. Reporter gene construct inserts are shown. (G) Luciferase reporter gene assay using the PPREs of the Ppara gene and its deleted-PPRE6 mutant, and Acox1 -PPRE repeat. n = 3 per group. Significant differences from cells without PPARA/RXRA expression plasmid or without Wy14,643: ∗ p < 0.001. (H) PPARA ChIP-qPCR assessed Ppara -PPRE6 or Acox1 -PPRE binding in liver samples from Ppara +/+ and Ppara −/- mice treated with Wy14,643. Mice fed either a control chow diet or a diet containing 0.1% Wy14,643 for 48 h. n = 3 mice per group. Significant differences from Wy14,643-treated Ppara +/+ mouse livers: ∗ p < 0.05. Each data point represents the mean ± SEM.

Article Snippet: pCMV3-mouse CDH1 , Sino Biological , Cat# MG50671-CF.

Techniques: Quantitative RT-PCR, Western Blot, ChIP-sequencing, Construct, Luciferase, Reporter Gene Assay, Mutagenesis, Expressing, Plasmid Preparation, Binding Assay

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet: Hepatocyte hyperproliferation by ligand-activated PPARA is suppressed by the forced expression of CDH1 (A and B) Western blot analysis (A) and the quantification (B) of CDH1 and GFP proteins in pCMV3-GFP (Control) or pCMV3-CDH1 (CDH1) plasmids-injected mice treated with Vehicle or Wy14,643. n = 3 mice per group. (C–E) H&E, CDH1-IHC, and BrdU-IHC staining (C), BrdU positive cells (D), and liver/body weight (E) in pCMV3-GFP (Control) or pCMV3-CDH1 (CDH1) plasmids-injected mice treated with Vehicle or Wy14,643. Scale bars represent 100 μm (200X). (F) qRT-PCR analysis of Myc , Ccnd1 , and Jun mRNAs in livers of pCMV3-GFP (Control) or pCMV3-CDH1 (CDH1) plasmids-injected mice treated with Vehicle or Wy14,643. Mice were treated with Wy14,643 (50 mg/12 h/kg for 36 h) or vehicle by oral gavage after injection each plasmid. Each data point represents the mean ± SEM n = 3–4 mice per group. Significant differences from normal chow treated Vehicle-Control-mouse livers or Wy14,643-Control-mouse livers: ∗ p < 0.05, ∗∗ p < 0.01. (G) Schematic of the mechanism by which mouse PPARA controls hepatocyte proliferation through the E2F8-UHRF1-CDH1 axis.

Article Snippet: pCMV3-mouse CDH1 , Sino Biological , Cat# MG50671-CF.

Techniques: Expressing, Western Blot, Injection, Immunohistochemistry, Quantitative RT-PCR, Plasmid Preparation

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet:

Article Snippet: pCMV3-mouse CDH1 , Sino Biological , Cat# MG50671-CF.

Techniques: Recombinant, Chromatin Immunoprecipitation, AST Assay, Immunoprecipitation, shRNA, Luciferase, Software