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Figure 2. Effects of PRH manipulation on CCA cell biology. A, Proliferation of CCLP1 cells stably transfected with PRH shRNA or scrambled control; n ¼ 3; P ¼ 0.03. B, Proliferation of CCLP1 and CCSW1 cells infected with Ad myc-PRH or empty virus control; n ¼ 3; P ¼ 0.03 (CCLP1); P ¼ 0.02 (CCSW1). C, Proliferation of AKN1 and primary BECs overexpressing GFP-PRH-myc (stable) or myc-PRH (transient, 48 hours); n ¼ 3; P ¼ 0.003 (AKN1); P ¼ 0.006 (BEC). D, Morphology of CCLP1 cells stably transfected with PRH shRNA or scrambled control. E, Western blot showing increased expression <t>of</t> <t>E-cadherin</t> protein and decreased expression of vimentin protein after PRH knockdown. Lamin A/C was used as loading control. F, Migration of CCLP1 PRH knockdown cells through transwell ﬁlters in a 10% serum gradient; n ¼ 3; P ¼ 0.03. G, Invasion of CCLP1 cells through Matrigel; n ¼ 4; P ¼ 0.03. H, Western blotting for myc-PRH and EMT-associated proteins E-cadherin and vimentin in AKN1 cells and primary BECs. I and J, As in F and G for AKN1 cells and primary BECs. K, Morphology of equal numbers of AKN1 cells stably transfected with plasmids expressing GFP (control) or a GFP-PRH-Myc–tagged fusion protein (GFP-PRH-Myc). Scale bars, 50 mm in length. , P < 0.05.

Cdh1 Cdna In Pcdna3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition"

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

Journal: Cancer Research

doi: 10.1158/0008-5472.can-19-0942

Figure Legend Snippet: Figure 2. Effects of PRH manipulation on CCA cell biology. A, Proliferation of CCLP1 cells stably transfected with PRH shRNA or scrambled control; n ¼ 3; P ¼ 0.03. B, Proliferation of CCLP1 and CCSW1 cells infected with Ad myc-PRH or empty virus control; n ¼ 3; P ¼ 0.03 (CCLP1); P ¼ 0.02 (CCSW1). C, Proliferation of AKN1 and primary BECs overexpressing GFP-PRH-myc (stable) or myc-PRH (transient, 48 hours); n ¼ 3; P ¼ 0.003 (AKN1); P ¼ 0.006 (BEC). D, Morphology of CCLP1 cells stably transfected with PRH shRNA or scrambled control. E, Western blot showing increased expression of E-cadherin protein and decreased expression of vimentin protein after PRH knockdown. Lamin A/C was used as loading control. F, Migration of CCLP1 PRH knockdown cells through transwell ﬁlters in a 10% serum gradient; n ¼ 3; P ¼ 0.03. G, Invasion of CCLP1 cells through Matrigel; n ¼ 4; P ¼ 0.03. H, Western blotting for myc-PRH and EMT-associated proteins E-cadherin and vimentin in AKN1 cells and primary BECs. I and J, As in F and G for AKN1 cells and primary BECs. K, Morphology of equal numbers of AKN1 cells stably transfected with plasmids expressing GFP (control) or a GFP-PRH-Myc–tagged fusion protein (GFP-PRH-Myc). Scale bars, 50 mm in length. , P < 0.05.

Techniques Used: Stable Transfection, Transfection, shRNA, Control, Infection, Virus, Western Blot, Expressing, Knockdown, Migration

Figure 4. Notch3 expression is regulated by PRH. A, Western blot showing Notch3 ICD protein expression in PRH knockdown CCLP1 cells. B, NOTCH3 gene expression in CCLP1 and CCSW1 cells infected with Ad myc-PRH, Ad myc-PRH DNA-binding deﬁcient N187A mutant, or empty virus control. C, Western blot for CCLP1 samples in B. D, Notch3 ICD rotein expression correlates with PRH protein expression in four human CCA cell lines, two human primary BEC isolates, and an immortalized BEC line. E, Western blot showing elevated expression of Notch3 ICD upon PRH overexpression in both AKN1 cells and primary BECs. F, Western blot showing increased expression of E-cadherin and reduced expression of PRH proteins after Notch3 knockdown. Lamin A/C was used as loading control. G, Proliferation of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control; n ¼ 3; P ¼ 0.04. H, Morphology of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control. I, DEGs detected in Notch3 KD compared with PRH KD experiments. Hypergeometric test, P ¼ 10229 for upregulated genes and P ¼ 1031 for downregulated genes. J, Hallmark GSEA of genes differentially expressed after Notch3 KD. Red bars, gene sets that are also enriched after PRH knockdown. , P < 0.05.

Figure Legend Snippet: Figure 4. Notch3 expression is regulated by PRH. A, Western blot showing Notch3 ICD protein expression in PRH knockdown CCLP1 cells. B, NOTCH3 gene expression in CCLP1 and CCSW1 cells infected with Ad myc-PRH, Ad myc-PRH DNA-binding deﬁcient N187A mutant, or empty virus control. C, Western blot for CCLP1 samples in B. D, Notch3 ICD rotein expression correlates with PRH protein expression in four human CCA cell lines, two human primary BEC isolates, and an immortalized BEC line. E, Western blot showing elevated expression of Notch3 ICD upon PRH overexpression in both AKN1 cells and primary BECs. F, Western blot showing increased expression of E-cadherin and reduced expression of PRH proteins after Notch3 knockdown. Lamin A/C was used as loading control. G, Proliferation of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control; n ¼ 3; P ¼ 0.04. H, Morphology of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control. I, DEGs detected in Notch3 KD compared with PRH KD experiments. Hypergeometric test, P ¼ 10229 for upregulated genes and P ¼ 1031 for downregulated genes. J, Hallmark GSEA of genes differentially expressed after Notch3 KD. Red bars, gene sets that are also enriched after PRH knockdown. , P < 0.05.

Techniques Used: Expressing, Western Blot, Knockdown, Gene Expression, Infection, Binding Assay, Mutagenesis, Virus, Control, Over Expression, Stable Transfection, Transfection, shRNA

Figure 5. Notch3- and PRH-correlated gene sets. A, qRT-PCR analysis of genes from CCLP1 cells overexpressing PRH in the presence or absence of Notch3 shRNA identifying PRH and Notch3-correlated expression signatures. B, Western blot analysis of EMT proteins E-cadherin and vimentin and cyclin D2 in CCLP1 cells overexpressing myc-PRH in the presence of absence of Notch3 shRNA. C, Proliferation of CCLP1 cells overexpressing myc-PRH in the presence or absence of Notch3 shRNA. , P < 0.05 after Bonferroni correction, compared with nontargeting shRNA/empty virus control. #, no statistically signiﬁcant difference in the comparison indicated. D, Hallmark GSEA of Notch3-correlated and PRH-correlated gene sets identiﬁed from analysis of RNA-seq data. Red bars, gene sets enriched in both PRH- and Notch3-correlated sets. E, Overlap of genes with PRH-binding sites determined by ChIP-seq and genes that are differentially expressed after PRH overexpression determined by RNA-seq in CCLP1 cells. F, Comparison of the primary motif underlying PRH ChIP-seq peaks identiﬁed using HOMER with the PRH SELEX motif (derived from ref. 38). G, RNA-seq and ChIP-seq tracks of putative direct PRH target DKK1. Red tracks, myc-PRH overexpression. H, RNA-seq and ChIP-seq tracks of putative direct PRH target WNT11. Red tracks, myc-PRH overexpression.

Figure Legend Snippet: Figure 5. Notch3- and PRH-correlated gene sets. A, qRT-PCR analysis of genes from CCLP1 cells overexpressing PRH in the presence or absence of Notch3 shRNA identifying PRH and Notch3-correlated expression signatures. B, Western blot analysis of EMT proteins E-cadherin and vimentin and cyclin D2 in CCLP1 cells overexpressing myc-PRH in the presence of absence of Notch3 shRNA. C, Proliferation of CCLP1 cells overexpressing myc-PRH in the presence or absence of Notch3 shRNA. , P < 0.05 after Bonferroni correction, compared with nontargeting shRNA/empty virus control. #, no statistically signiﬁcant difference in the comparison indicated. D, Hallmark GSEA of Notch3-correlated and PRH-correlated gene sets identiﬁed from analysis of RNA-seq data. Red bars, gene sets enriched in both PRH- and Notch3-correlated sets. E, Overlap of genes with PRH-binding sites determined by ChIP-seq and genes that are differentially expressed after PRH overexpression determined by RNA-seq in CCLP1 cells. F, Comparison of the primary motif underlying PRH ChIP-seq peaks identiﬁed using HOMER with the PRH SELEX motif (derived from ref. 38). G, RNA-seq and ChIP-seq tracks of putative direct PRH target DKK1. Red tracks, myc-PRH overexpression. H, RNA-seq and ChIP-seq tracks of putative direct PRH target WNT11. Red tracks, myc-PRH overexpression.

Techniques Used: Quantitative RT-PCR, shRNA, Expressing, Western Blot, Virus, Control, Comparison, RNA Sequencing, Binding Assay, ChIP-sequencing, Over Expression, Derivative Assay

$Figure 6. Regulation of Wnt signaling. A, TOPFlash TCF/LEF reporter activity in CCLP1 and CCSW1 cells expressing myc-PRH, DNA-binding–deﬁcient N187A mutant of myc- PRH and Flag-Notch3-ICD. Inset, Western blot showing expression of myc-PRH constructs. B, Representative immunoﬂuorescence micrographs of CCLP1 PRH knockdown cells stained for E-cadherin and b-catenin. C, Western blot of subcellular fractions of CCLP1 PRH knockdown cells. D, TOPFlash reporter activity in PRH knockdown CCLP1 cells. E, TOPFlash reporter activity in control CCLP1 cells (pcDNA empty) and CCLP1 cells overexpressing E-cadherin (CDH1) in the presence and absence of myc-PRH expression. Inset, Western blot showing successful overexpression of E-cadherin. , P < 0.05; , P < 0.01; #, no statistically signiﬁcant difference.$
Figure Legend Snippet: Figure 6. Regulation of Wnt signaling. A, TOPFlash TCF/LEF reporter activity in CCLP1 and CCSW1 cells expressing myc-PRH, DNA-binding–deﬁcient N187A mutant of myc- PRH and Flag-Notch3-ICD. Inset, Western blot showing expression of myc-PRH constructs. B, Representative immunoﬂuorescence micrographs of CCLP1 PRH knockdown cells stained for E-cadherin and b-catenin. C, Western blot of subcellular fractions of CCLP1 PRH knockdown cells. D, TOPFlash reporter activity in PRH knockdown CCLP1 cells. E, TOPFlash reporter activity in control CCLP1 cells (pcDNA empty) and CCLP1 cells overexpressing E-cadherin (CDH1) in the presence and absence of myc-PRH expression. Inset, Western blot showing successful overexpression of E-cadherin. , P < 0.05; , P < 0.01; #, no statistically signiﬁcant difference.

Techniques Used: Activity Assay, Expressing, Binding Assay, Mutagenesis, Western Blot, Construct, Knockdown, Staining, Control, Over Expression

Image Search Results

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.

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

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.

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

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.

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

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.

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

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.

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

Journal: iScience

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

doi: 10.1016/j.isci.2022.104196

Article Snippet: C57BL/6J mice were tail-vein injected with pCMV3- CDH1 (Sino Biological, mouse/MG50671-CF, ) or EGFP (Sino Biological, Aequorea Victoria/AG13105-CF) using PEI Max transfection reagent (PEI, Polysciences) according to a method modified from .

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

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

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

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

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:

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

Journal: Cancer Research

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

doi: 10.1158/0008-5472.can-19-0942

Figure Lengend Snippet: Figure 2. Effects of PRH manipulation on CCA cell biology. A, Proliferation of CCLP1 cells stably transfected with PRH shRNA or scrambled control; n ¼ 3; P ¼ 0.03. B, Proliferation of CCLP1 and CCSW1 cells infected with Ad myc-PRH or empty virus control; n ¼ 3; P ¼ 0.03 (CCLP1); P ¼ 0.02 (CCSW1). C, Proliferation of AKN1 and primary BECs overexpressing GFP-PRH-myc (stable) or myc-PRH (transient, 48 hours); n ¼ 3; P ¼ 0.003 (AKN1); P ¼ 0.006 (BEC). D, Morphology of CCLP1 cells stably transfected with PRH shRNA or scrambled control. E, Western blot showing increased expression of E-cadherin protein and decreased expression of vimentin protein after PRH knockdown. Lamin A/C was used as loading control. F, Migration of CCLP1 PRH knockdown cells through transwell ﬁlters in a 10% serum gradient; n ¼ 3; P ¼ 0.03. G, Invasion of CCLP1 cells through Matrigel; n ¼ 4; P ¼ 0.03. H, Western blotting for myc-PRH and EMT-associated proteins E-cadherin and vimentin in AKN1 cells and primary BECs. I and J, As in F and G for AKN1 cells and primary BECs. K, Morphology of equal numbers of AKN1 cells stably transfected with plasmids expressing GFP (control) or a GFP-PRH-Myc–tagged fusion protein (GFP-PRH-Myc). Scale bars, 50 mm in length. , P < 0.05.

Article Snippet: Stable cell lines were generated by transfecting PRH shRNA in pRS (Origene, TR312464), Notch3 shRNA in pLKO (Sigma-Aldrich, TRCN0000363316), CDH1 cDNA in pCDNA3 (hE-cadherinpcDNA3 was a gift from Dr. Barry Gumbiner, Seattle Children's Hospital, Seattle, WA; Addgene plasmid #45769), or EGFP-PRHmyc in pEGFP-C1.

Techniques: Stable Transfection, Transfection, shRNA, Control, Infection, Virus, Western Blot, Expressing, Knockdown, Migration

Journal: Cancer Research

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

doi: 10.1158/0008-5472.can-19-0942

Figure Lengend Snippet: Figure 4. Notch3 expression is regulated by PRH. A, Western blot showing Notch3 ICD protein expression in PRH knockdown CCLP1 cells. B, NOTCH3 gene expression in CCLP1 and CCSW1 cells infected with Ad myc-PRH, Ad myc-PRH DNA-binding deﬁcient N187A mutant, or empty virus control. C, Western blot for CCLP1 samples in B. D, Notch3 ICD rotein expression correlates with PRH protein expression in four human CCA cell lines, two human primary BEC isolates, and an immortalized BEC line. E, Western blot showing elevated expression of Notch3 ICD upon PRH overexpression in both AKN1 cells and primary BECs. F, Western blot showing increased expression of E-cadherin and reduced expression of PRH proteins after Notch3 knockdown. Lamin A/C was used as loading control. G, Proliferation of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control; n ¼ 3; P ¼ 0.04. H, Morphology of CCLP1 cells stably transfected with Notch3 shRNA or scrambled control. I, DEGs detected in Notch3 KD compared with PRH KD experiments. Hypergeometric test, P ¼ 10229 for upregulated genes and P ¼ 1031 for downregulated genes. J, Hallmark GSEA of genes differentially expressed after Notch3 KD. Red bars, gene sets that are also enriched after PRH knockdown. , P < 0.05.

Techniques: Expressing, Western Blot, Knockdown, Gene Expression, Infection, Binding Assay, Mutagenesis, Virus, Control, Over Expression, Stable Transfection, Transfection, shRNA

Journal: Cancer Research

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

doi: 10.1158/0008-5472.can-19-0942

Figure Lengend Snippet: Figure 5. Notch3- and PRH-correlated gene sets. A, qRT-PCR analysis of genes from CCLP1 cells overexpressing PRH in the presence or absence of Notch3 shRNA identifying PRH and Notch3-correlated expression signatures. B, Western blot analysis of EMT proteins E-cadherin and vimentin and cyclin D2 in CCLP1 cells overexpressing myc-PRH in the presence of absence of Notch3 shRNA. C, Proliferation of CCLP1 cells overexpressing myc-PRH in the presence or absence of Notch3 shRNA. , P < 0.05 after Bonferroni correction, compared with nontargeting shRNA/empty virus control. #, no statistically signiﬁcant difference in the comparison indicated. D, Hallmark GSEA of Notch3-correlated and PRH-correlated gene sets identiﬁed from analysis of RNA-seq data. Red bars, gene sets enriched in both PRH- and Notch3-correlated sets. E, Overlap of genes with PRH-binding sites determined by ChIP-seq and genes that are differentially expressed after PRH overexpression determined by RNA-seq in CCLP1 cells. F, Comparison of the primary motif underlying PRH ChIP-seq peaks identiﬁed using HOMER with the PRH SELEX motif (derived from ref. 38). G, RNA-seq and ChIP-seq tracks of putative direct PRH target DKK1. Red tracks, myc-PRH overexpression. H, RNA-seq and ChIP-seq tracks of putative direct PRH target WNT11. Red tracks, myc-PRH overexpression.

Techniques: Quantitative RT-PCR, shRNA, Expressing, Western Blot, Virus, Control, Comparison, RNA Sequencing, Binding Assay, ChIP-sequencing, Over Expression, Derivative Assay

Journal: Cancer Research

Article Title: A Runaway PRH/HHEX-Notch3–Positive Feedback Loop Drives Cholangiocarcinoma and Determines Response to CDK4/6 Inhibition

doi: 10.1158/0008-5472.can-19-0942

Figure Lengend Snippet: Figure 6. Regulation of Wnt signaling. A, TOPFlash TCF/LEF reporter activity in CCLP1 and CCSW1 cells expressing myc-PRH, DNA-binding–deﬁcient N187A mutant of myc- PRH and Flag-Notch3-ICD. Inset, Western blot showing expression of myc-PRH constructs. B, Representative immunoﬂuorescence micrographs of CCLP1 PRH knockdown cells stained for E-cadherin and b-catenin. C, Western blot of subcellular fractions of CCLP1 PRH knockdown cells. D, TOPFlash reporter activity in PRH knockdown CCLP1 cells. E, TOPFlash reporter activity in control CCLP1 cells (pcDNA empty) and CCLP1 cells overexpressing E-cadherin (CDH1) in the presence and absence of myc-PRH expression. Inset, Western blot showing successful overexpression of E-cadherin. , P < 0.05; , P < 0.01; #, no statistically signiﬁcant difference.

Techniques: Activity Assay, Expressing, Binding Assay, Mutagenesis, Western Blot, Construct, Knockdown, Staining, Control, Over Expression

(A) Luciferase reporter plasmids containing the putative wild-type or mutant miR-23a binding sequence in 3’-UTR of CDH1 mRNA. (B) HEK293T cells were co-transfected with a control vector or miR-23a and a luciferase reporter construct containing the wild-type or mutant CDH1 3’-UTR. The results were normalized, and the luciferase activity of the control was set to 1. (C and D) Effects of miR-23a dysregulation of CDH1 expression were detected by western blot analysis in MCF-7 and MDA-MB-231 cells. (E) Real-time PCR analysis of CDH1 mRNA expression of the NC and anti-miR-23a-transfected cells treated with or without TGF-β1. *P<0.05, **P<0.01.

Journal: Oncotarget

Article Title: MiR-23a promotes TGF-β1-induced EMT and tumor metastasis in breast cancer cells by directly targeting CDH1 and activating Wnt/β-catenin signaling

doi: 10.18632/oncotarget.18422

Figure Lengend Snippet: (A) Luciferase reporter plasmids containing the putative wild-type or mutant miR-23a binding sequence in 3’-UTR of CDH1 mRNA. (B) HEK293T cells were co-transfected with a control vector or miR-23a and a luciferase reporter construct containing the wild-type or mutant CDH1 3’-UTR. The results were normalized, and the luciferase activity of the control was set to 1. (C and D) Effects of miR-23a dysregulation of CDH1 expression were detected by western blot analysis in MCF-7 and MDA-MB-231 cells. (E) Real-time PCR analysis of CDH1 mRNA expression of the NC and anti-miR-23a-transfected cells treated with or without TGF-β1. *P<0.05, **P<0.01.

Article Snippet: CDH1 Human cDNA ORF Clone was purchased from Origene (Origene Technologies).

Techniques: Luciferase, Mutagenesis, Binding Assay, Sequencing, Transfection, Control, Plasmid Preparation, Construct, Activity Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

(A) Western blot analysis confirmed the transfection of constructs containing CDH1 ORF in cells treated with TGF-β1. (B) Western blot analysis confirmed the transfection of specific siRNA in miR-23a-silenced cells treated with TGF-β1. (C) Reintroduction of E-cadherin in TGF-β1-treated MDA-MB-231 cells abrogated TGF-β1-induced cell invasion. MiR-23a inhibitor reduced the invasion and migration cells treated by TGF-β1 and siCDH1 restored the cell invasion and migration in miR-23a-silenced cells. (D) Tumor metastasis analysis. The number of metastatic lung nodules in each group of nude mice (n= 6 per group, left panel). Representative photos of nude mice injected with indicated cells and micrographs of HE staining of metastatic tumor tissues (right panel). *P<0.05, **P<0.01.

Journal: Oncotarget

Article Title: MiR-23a promotes TGF-β1-induced EMT and tumor metastasis in breast cancer cells by directly targeting CDH1 and activating Wnt/β-catenin signaling

doi: 10.18632/oncotarget.18422

Figure Lengend Snippet: (A) Western blot analysis confirmed the transfection of constructs containing CDH1 ORF in cells treated with TGF-β1. (B) Western blot analysis confirmed the transfection of specific siRNA in miR-23a-silenced cells treated with TGF-β1. (C) Reintroduction of E-cadherin in TGF-β1-treated MDA-MB-231 cells abrogated TGF-β1-induced cell invasion. MiR-23a inhibitor reduced the invasion and migration cells treated by TGF-β1 and siCDH1 restored the cell invasion and migration in miR-23a-silenced cells. (D) Tumor metastasis analysis. The number of metastatic lung nodules in each group of nude mice (n= 6 per group, left panel). Representative photos of nude mice injected with indicated cells and micrographs of HE staining of metastatic tumor tissues (right panel). *P<0.05, **P<0.01.

Article Snippet: CDH1 Human cDNA ORF Clone was purchased from Origene (Origene Technologies).

Techniques: Western Blot, Transfection, Construct, Migration, Injection, Staining

$(A) Nuclear fraction of indicated cells was analyzed by Western blot. Lamin B1 was used as a loading control. (B) Beta-catenin localization in indicated cells was detected by immunofluorescence staining. (C) Indicated cells were transfected with TOPflash and Renilla pRL-TK plasmids, and subjected to dual –luciferase assays 48h after transfection. Reporter activity was normalized to Renilla luciferase activity. (D and E) Transwell assays were used to detect the quantification of invading cells transfected with indicated siRNAs and luciferase-reported TCF/LEF transcriptional activity in indicated cells were examined. (F) Western blot analysis was used to confirm the transfection of constructs containing CDH1 ORF in miR-23a overexpressed MCF-7 cells (left panel). Quantification of invading cells transfected with indicated construct (middle panel) and luciferase-reported TCF/LEF transcriptional activity in indicated cells (right panel). (G) Luciferase-reported TCF/LEF transcriptional activity in NC, cells treated with TGF-β1 or miR-23a silenced-cells treated with TGF-β1. (H) Schematic representation of a model for the role of miR-23a in the TGF-β-induced tumor metastasis in breast cancer. *P<0.05, **P<0.01.$

Journal: Oncotarget

Article Title: MiR-23a promotes TGF-β1-induced EMT and tumor metastasis in breast cancer cells by directly targeting CDH1 and activating Wnt/β-catenin signaling

doi: 10.18632/oncotarget.18422

Figure Lengend Snippet: (A) Nuclear fraction of indicated cells was analyzed by Western blot. Lamin B1 was used as a loading control. (B) Beta-catenin localization in indicated cells was detected by immunofluorescence staining. (C) Indicated cells were transfected with TOPflash and Renilla pRL-TK plasmids, and subjected to dual –luciferase assays 48h after transfection. Reporter activity was normalized to Renilla luciferase activity. (D and E) Transwell assays were used to detect the quantification of invading cells transfected with indicated siRNAs and luciferase-reported TCF/LEF transcriptional activity in indicated cells were examined. (F) Western blot analysis was used to confirm the transfection of constructs containing CDH1 ORF in miR-23a overexpressed MCF-7 cells (left panel). Quantification of invading cells transfected with indicated construct (middle panel) and luciferase-reported TCF/LEF transcriptional activity in indicated cells (right panel). (G) Luciferase-reported TCF/LEF transcriptional activity in NC, cells treated with TGF-β1 or miR-23a silenced-cells treated with TGF-β1. (H) Schematic representation of a model for the role of miR-23a in the TGF-β-induced tumor metastasis in breast cancer. *P<0.05, **P<0.01.

Article Snippet: CDH1 Human cDNA ORF Clone was purchased from Origene (Origene Technologies).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Transfection, Luciferase, Activity Assay, Construct

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