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Santa Cruz Biotechnology mouse anti-human e2f2 antibody

The overexpression of C/EBPβ promoted the proliferation of HPCs. ( A ) The diagram shows that cells were disassociated by TrypLE at HPC D7 and then seeded at 4 × 10 4 cells/cm 2 on collagen type I coated plates. Simultaneously, PLV-C/EBPβ virus was administrated. Subsequently, cells were disassociated again and counted at HPC D8 and HPC D14, respectively. ( B ) Cell numbers were determined by cell counts for HPC on day 8 and for HPC on day 14. ( C ) GSEA results showed that cell cycle and E2F targets related genes were significantly enriched in HPCs transduced with PLV-C/EBPβ on day 14 when compared with those transduced with PLV-Vector on day 14. ( D ) Heat maps of selected proliferative genes and suppressor genes were differentially expressed between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. ( E ) The KEGG pathways for the differentially expressed genes between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. The principal pathways were the cell cycle and DNA replication. ( F ) Volcano plots showed significant changes in cell-cycle-related genes between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. Volcano plot showed −log10 ( p -value) on the y-axis versus log2 (fold change) on the x-axis. Each point represented a different gene. ( G , H ) Relative expression of proliferative genes, including PCNA, CDC25C, CDC45L, MCM3, GINS1, and CCND1 ( F ) and E2F genes, including E2F1 and <t>E2F2</t> ( H ) in HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.001 ***, ns, not significant. Data are the three samples per group.

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1) Product Images from "C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes"

Article Title: C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes

Journal: Cells

doi: 10.3390/cells12030497

Figure Legend Snippet: The overexpression of C/EBPβ promoted the proliferation of HPCs. ( A ) The diagram shows that cells were disassociated by TrypLE at HPC D7 and then seeded at 4 × 10 4 cells/cm 2 on collagen type I coated plates. Simultaneously, PLV-C/EBPβ virus was administrated. Subsequently, cells were disassociated again and counted at HPC D8 and HPC D14, respectively. ( B ) Cell numbers were determined by cell counts for HPC on day 8 and for HPC on day 14. ( C ) GSEA results showed that cell cycle and E2F targets related genes were significantly enriched in HPCs transduced with PLV-C/EBPβ on day 14 when compared with those transduced with PLV-Vector on day 14. ( D ) Heat maps of selected proliferative genes and suppressor genes were differentially expressed between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. ( E ) The KEGG pathways for the differentially expressed genes between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. The principal pathways were the cell cycle and DNA replication. ( F ) Volcano plots showed significant changes in cell-cycle-related genes between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. Volcano plot showed −log10 ( p -value) on the y-axis versus log2 (fold change) on the x-axis. Each point represented a different gene. ( G , H ) Relative expression of proliferative genes, including PCNA, CDC25C, CDC45L, MCM3, GINS1, and CCND1 ( F ) and E2F genes, including E2F1 and E2F2 ( H ) in HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.001 ***, ns, not significant. Data are the three samples per group.

Techniques Used: Over Expression, Transduction, Plasmid Preparation, Expressing

C/EBPβ Knockdown impaired hepatic differentiation and blocked proliferation of HPCs. ( A ) Representative images of cell morphologies of HPCs 2 days after si-RNA treatments on day 12 and cell numbers were counted in each group in ( A ). ( B ) Schematic illustration of si-RNA mediated C/EBPβ knockdown during the hepatic differentiation of hESCs. ( C ) Knockdown efficiency of C/EBPβ was detected by qRT-PCR on day 14 of HPCs. ( D ) ALB secretion was detected from the supernatants of cultured HPCs 24 h after siRNA (C/EBPβ) treatment. ( E ) Relative gene expression analysis of hepatocyte genes including AFP, ALB, ASGPR1, A1AT, HNF4A, and CEBPα in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( F ) Heat map of selected genes, including hepatocyte genes, proliferative genes, and suppressor genes in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( G ) The same number of LO2 cells were seeded, and then si-RNA mediated C/EBPβ knockdown was conducted. After 3 days of treatments, DAPI staining was performed to mark the nuclei, and then cell numbers were defined by ImageJ software. ( H ) Relative expression of proliferative genes including CDC25C, CDC45L, GINS1, MCM3, E2F1, and E2F2 in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( I ) GSEA results showed that cell-cycle-related gene sets, such as MYC targets, cell-cycle literature, and mitosis targets bound by E2F, were significantly enriched in HPCs treated with si-NC when compared with those in HPCs treated with si-C/EBPβ. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.0001 ****, ns, not significant. Data are the three samples per group.

Figure Legend Snippet: C/EBPβ Knockdown impaired hepatic differentiation and blocked proliferation of HPCs. ( A ) Representative images of cell morphologies of HPCs 2 days after si-RNA treatments on day 12 and cell numbers were counted in each group in ( A ). ( B ) Schematic illustration of si-RNA mediated C/EBPβ knockdown during the hepatic differentiation of hESCs. ( C ) Knockdown efficiency of C/EBPβ was detected by qRT-PCR on day 14 of HPCs. ( D ) ALB secretion was detected from the supernatants of cultured HPCs 24 h after siRNA (C/EBPβ) treatment. ( E ) Relative gene expression analysis of hepatocyte genes including AFP, ALB, ASGPR1, A1AT, HNF4A, and CEBPα in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( F ) Heat map of selected genes, including hepatocyte genes, proliferative genes, and suppressor genes in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( G ) The same number of LO2 cells were seeded, and then si-RNA mediated C/EBPβ knockdown was conducted. After 3 days of treatments, DAPI staining was performed to mark the nuclei, and then cell numbers were defined by ImageJ software. ( H ) Relative expression of proliferative genes including CDC25C, CDC45L, GINS1, MCM3, E2F1, and E2F2 in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( I ) GSEA results showed that cell-cycle-related gene sets, such as MYC targets, cell-cycle literature, and mitosis targets bound by E2F, were significantly enriched in HPCs treated with si-NC when compared with those in HPCs treated with si-C/EBPβ. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.0001 ****, ns, not significant. Data are the three samples per group.

Techniques Used: Quantitative RT-PCR, Cell Culture, Expressing, Staining, Software

C/EBPβ coupled with E2F2 orchestrated cell proliferation of HPCs. ( A , B ) Western blot analysis of C/EBPβ on day 14 of HPCs after the knockdown or overexpression of C/EBPβ ( A ), and the statistical results ( B ). ( C , D ) Western blot analysis of representative protein expressions associated with cell proliferation after knockdown of C/EBPβ (C) , and corresponding statistical results ( D ). ( E , F ) Western blot analysis of representative protein expressions associated with cell proliferation after overexpression of C/EBPβ ( E ) and corresponding statistical results ( F ). ( G , H ) Co-immunoprecipitation (Co-IP) assays to validate interaction of C/EBPβ with E2F2 in HPCs on day 14 and LO2 cells. The cell lysates were subjected to immunoprecipitation with C/EBPβ antibody, and the resulting immunoprecipitants were analyzed in the immunoblot with E2F2 and C/EBPβ antibodies (IB: E2F2; IB: C/EBPβ). Input was also subjected to immunoblot to represent a positive control, and non-specific IgG represented as a negative control. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, ns, not significant. Data are the three samples per group.

Figure Legend Snippet: C/EBPβ coupled with E2F2 orchestrated cell proliferation of HPCs. ( A , B ) Western blot analysis of C/EBPβ on day 14 of HPCs after the knockdown or overexpression of C/EBPβ ( A ), and the statistical results ( B ). ( C , D ) Western blot analysis of representative protein expressions associated with cell proliferation after knockdown of C/EBPβ (C) , and corresponding statistical results ( D ). ( E , F ) Western blot analysis of representative protein expressions associated with cell proliferation after overexpression of C/EBPβ ( E ) and corresponding statistical results ( F ). ( G , H ) Co-immunoprecipitation (Co-IP) assays to validate interaction of C/EBPβ with E2F2 in HPCs on day 14 and LO2 cells. The cell lysates were subjected to immunoprecipitation with C/EBPβ antibody, and the resulting immunoprecipitants were analyzed in the immunoblot with E2F2 and C/EBPβ antibodies (IB: E2F2; IB: C/EBPβ). Input was also subjected to immunoblot to represent a positive control, and non-specific IgG represented as a negative control. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, ns, not significant. Data are the three samples per group.

Techniques Used: Western Blot, Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay, Positive Control, Negative Control

C/EBPβ promoted the expression of proliferative genes by binding to promoter regions. ( A ) CUT&Tag data analysis for peak summit of relative genes at location map, the abscissa was the distance between peak summit and TSS site, and the ordinate was the number of peaks. ( B ) Pie chart of peak distribution on gene functional elements. ( C ) Top scoring and representative motif sequence of C/EBPβ binding sites. ( D ) KEGG enrichment bubble diagram to show the first 20 pathways with the smallest q values, pathway shown as the ordinate and rich factor shown as the abscissa (the number of differences in this pathway divided by all the numbers). The size indicated the number; the redder the color, the smaller the q value. ( E – I ) The peaks of individual proliferative genes, including CDC25C, CDC45L, PCNA, CDC7, and E2F2, were enriched in the transcriptional regulatory regions of their respective genes, and the peaks of some genes were enhanced after overexpression of C/EBPβ.

Figure Legend Snippet: C/EBPβ promoted the expression of proliferative genes by binding to promoter regions. ( A ) CUT&Tag data analysis for peak summit of relative genes at location map, the abscissa was the distance between peak summit and TSS site, and the ordinate was the number of peaks. ( B ) Pie chart of peak distribution on gene functional elements. ( C ) Top scoring and representative motif sequence of C/EBPβ binding sites. ( D ) KEGG enrichment bubble diagram to show the first 20 pathways with the smallest q values, pathway shown as the ordinate and rich factor shown as the abscissa (the number of differences in this pathway divided by all the numbers). The size indicated the number; the redder the color, the smaller the q value. ( E – I ) The peaks of individual proliferative genes, including CDC25C, CDC45L, PCNA, CDC7, and E2F2, were enriched in the transcriptional regulatory regions of their respective genes, and the peaks of some genes were enhanced after overexpression of C/EBPβ.

Techniques Used: Expressing, Binding Assay, Functional Assay, Sequencing, Over Expression

Cell-cycle progression was promoted by C/EBPβ in LO2 cells. ( A ) The schematic diagram shows that C/EBPβ binds to CDC45L promoter regions to regulate luciferase expression. ( B ) Luciferase assays were carried out to verify the C/EBPβ binding activity at promoter regions of CDC45L gene in 293T cells and LO2 cells. ( C , D ) Lentivirus-mediated C/EBPβ knockdown with three pairs of sh-RNA (sh-a, sh-b, and sh-c) was performed to determine the expression of cell-cycle genes ( C ) and transcriptional factor E2Fs genes ( D ) by qRT-PCR assays. ( E , F ) Flow cytometry was performed to detect the proportion of cell-cycle phases after the treatment with sh-RNA (C/EBPβ) in LO2 cells ( E ), and the statistical results of proportion were depicted ( F ) by GraphPad Prism 8 software. ( G ) C/EBPβ coupled with E2F2 regulated cell-cycle gene expression in hepatic progenitor cells. C/EBPβ first bound to the regulatory region of cell-cycle-regulating transcription factor E2F2 to activate E2F2, then C/EBPβ coupled with E2F2 to promote the expression of cell-cycle genes (CDC45L, CDC25C, and PCNA) in hepatic progenitor cells. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****, ns, not significant. Data are the three samples per group.

Figure Legend Snippet: Cell-cycle progression was promoted by C/EBPβ in LO2 cells. ( A ) The schematic diagram shows that C/EBPβ binds to CDC45L promoter regions to regulate luciferase expression. ( B ) Luciferase assays were carried out to verify the C/EBPβ binding activity at promoter regions of CDC45L gene in 293T cells and LO2 cells. ( C , D ) Lentivirus-mediated C/EBPβ knockdown with three pairs of sh-RNA (sh-a, sh-b, and sh-c) was performed to determine the expression of cell-cycle genes ( C ) and transcriptional factor E2Fs genes ( D ) by qRT-PCR assays. ( E , F ) Flow cytometry was performed to detect the proportion of cell-cycle phases after the treatment with sh-RNA (C/EBPβ) in LO2 cells ( E ), and the statistical results of proportion were depicted ( F ) by GraphPad Prism 8 software. ( G ) C/EBPβ coupled with E2F2 regulated cell-cycle gene expression in hepatic progenitor cells. C/EBPβ first bound to the regulatory region of cell-cycle-regulating transcription factor E2F2 to activate E2F2, then C/EBPβ coupled with E2F2 to promote the expression of cell-cycle genes (CDC45L, CDC25C, and PCNA) in hepatic progenitor cells. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****, ns, not significant. Data are the three samples per group.

Techniques Used: Luciferase, Expressing, Binding Assay, Activity Assay, Quantitative RT-PCR, Flow Cytometry, Software

Image Search Results

Journal: Cells

Article Title: C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes

doi: 10.3390/cells12030497

Figure Lengend Snippet: The overexpression of C/EBPβ promoted the proliferation of HPCs. ( A ) The diagram shows that cells were disassociated by TrypLE at HPC D7 and then seeded at 4 × 10 4 cells/cm 2 on collagen type I coated plates. Simultaneously, PLV-C/EBPβ virus was administrated. Subsequently, cells were disassociated again and counted at HPC D8 and HPC D14, respectively. ( B ) Cell numbers were determined by cell counts for HPC on day 8 and for HPC on day 14. ( C ) GSEA results showed that cell cycle and E2F targets related genes were significantly enriched in HPCs transduced with PLV-C/EBPβ on day 14 when compared with those transduced with PLV-Vector on day 14. ( D ) Heat maps of selected proliferative genes and suppressor genes were differentially expressed between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. ( E ) The KEGG pathways for the differentially expressed genes between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. The principal pathways were the cell cycle and DNA replication. ( F ) Volcano plots showed significant changes in cell-cycle-related genes between HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. Volcano plot showed −log10 ( p -value) on the y-axis versus log2 (fold change) on the x-axis. Each point represented a different gene. ( G , H ) Relative expression of proliferative genes, including PCNA, CDC25C, CDC45L, MCM3, GINS1, and CCND1 ( F ) and E2F genes, including E2F1 and E2F2 ( H ) in HPCs transduced with PLV-Vector or with PLV- C/EBPβ on day 14. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.001 ***, ns, not significant. Data are the three samples per group.

Article Snippet: Subsequently, the aforementioned protein complexes were eluted from the beads, and then, agarose gel electrophoresis and Western blot were performed to transfer target proteins to PVDF membranes, followed by incubating with mouse anti-human C/EBPβ antibody (1:1000, santa cruz) or mouse anti-human E2F2 antibody (1:500, santa cruz).

Techniques: Over Expression, Transduction, Plasmid Preparation, Expressing

Journal: Cells

Article Title: C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes

doi: 10.3390/cells12030497

Figure Lengend Snippet: C/EBPβ Knockdown impaired hepatic differentiation and blocked proliferation of HPCs. ( A ) Representative images of cell morphologies of HPCs 2 days after si-RNA treatments on day 12 and cell numbers were counted in each group in ( A ). ( B ) Schematic illustration of si-RNA mediated C/EBPβ knockdown during the hepatic differentiation of hESCs. ( C ) Knockdown efficiency of C/EBPβ was detected by qRT-PCR on day 14 of HPCs. ( D ) ALB secretion was detected from the supernatants of cultured HPCs 24 h after siRNA (C/EBPβ) treatment. ( E ) Relative gene expression analysis of hepatocyte genes including AFP, ALB, ASGPR1, A1AT, HNF4A, and CEBPα in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( F ) Heat map of selected genes, including hepatocyte genes, proliferative genes, and suppressor genes in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( G ) The same number of LO2 cells were seeded, and then si-RNA mediated C/EBPβ knockdown was conducted. After 3 days of treatments, DAPI staining was performed to mark the nuclei, and then cell numbers were defined by ImageJ software. ( H ) Relative expression of proliferative genes including CDC25C, CDC45L, GINS1, MCM3, E2F1, and E2F2 in HPCs treated with si-NC or with si-C/EBPβ on day 14. ( I ) GSEA results showed that cell-cycle-related gene sets, such as MYC targets, cell-cycle literature, and mitosis targets bound by E2F, were significantly enriched in HPCs treated with si-NC when compared with those in HPCs treated with si-C/EBPβ. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.0001 ****, ns, not significant. Data are the three samples per group.

Techniques: Quantitative RT-PCR, Cell Culture, Expressing, Staining, Software

Journal: Cells

Article Title: C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes

doi: 10.3390/cells12030497

Figure Lengend Snippet: C/EBPβ coupled with E2F2 orchestrated cell proliferation of HPCs. ( A , B ) Western blot analysis of C/EBPβ on day 14 of HPCs after the knockdown or overexpression of C/EBPβ ( A ), and the statistical results ( B ). ( C , D ) Western blot analysis of representative protein expressions associated with cell proliferation after knockdown of C/EBPβ (C) , and corresponding statistical results ( D ). ( E , F ) Western blot analysis of representative protein expressions associated with cell proliferation after overexpression of C/EBPβ ( E ) and corresponding statistical results ( F ). ( G , H ) Co-immunoprecipitation (Co-IP) assays to validate interaction of C/EBPβ with E2F2 in HPCs on day 14 and LO2 cells. The cell lysates were subjected to immunoprecipitation with C/EBPβ antibody, and the resulting immunoprecipitants were analyzed in the immunoblot with E2F2 and C/EBPβ antibodies (IB: E2F2; IB: C/EBPβ). Input was also subjected to immunoblot to represent a positive control, and non-specific IgG represented as a negative control. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, ns, not significant. Data are the three samples per group.

Techniques: Western Blot, Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay, Positive Control, Negative Control

Journal: Cells

Article Title: C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes

doi: 10.3390/cells12030497

Figure Lengend Snippet: C/EBPβ promoted the expression of proliferative genes by binding to promoter regions. ( A ) CUT&Tag data analysis for peak summit of relative genes at location map, the abscissa was the distance between peak summit and TSS site, and the ordinate was the number of peaks. ( B ) Pie chart of peak distribution on gene functional elements. ( C ) Top scoring and representative motif sequence of C/EBPβ binding sites. ( D ) KEGG enrichment bubble diagram to show the first 20 pathways with the smallest q values, pathway shown as the ordinate and rich factor shown as the abscissa (the number of differences in this pathway divided by all the numbers). The size indicated the number; the redder the color, the smaller the q value. ( E – I ) The peaks of individual proliferative genes, including CDC25C, CDC45L, PCNA, CDC7, and E2F2, were enriched in the transcriptional regulatory regions of their respective genes, and the peaks of some genes were enhanced after overexpression of C/EBPβ.

Techniques: Expressing, Binding Assay, Functional Assay, Sequencing, Over Expression

Journal: Cells

Article Title: C/EBPβ Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes

doi: 10.3390/cells12030497

Figure Lengend Snippet: Cell-cycle progression was promoted by C/EBPβ in LO2 cells. ( A ) The schematic diagram shows that C/EBPβ binds to CDC45L promoter regions to regulate luciferase expression. ( B ) Luciferase assays were carried out to verify the C/EBPβ binding activity at promoter regions of CDC45L gene in 293T cells and LO2 cells. ( C , D ) Lentivirus-mediated C/EBPβ knockdown with three pairs of sh-RNA (sh-a, sh-b, and sh-c) was performed to determine the expression of cell-cycle genes ( C ) and transcriptional factor E2Fs genes ( D ) by qRT-PCR assays. ( E , F ) Flow cytometry was performed to detect the proportion of cell-cycle phases after the treatment with sh-RNA (C/EBPβ) in LO2 cells ( E ), and the statistical results of proportion were depicted ( F ) by GraphPad Prism 8 software. ( G ) C/EBPβ coupled with E2F2 regulated cell-cycle gene expression in hepatic progenitor cells. C/EBPβ first bound to the regulatory region of cell-cycle-regulating transcription factor E2F2 to activate E2F2, then C/EBPβ coupled with E2F2 to promote the expression of cell-cycle genes (CDC45L, CDC25C, and PCNA) in hepatic progenitor cells. Data represent mean ± SEM with p -values indicated when significant. p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****, ns, not significant. Data are the three samples per group.

Techniques: Luciferase, Expressing, Binding Assay, Activity Assay, Quantitative RT-PCR, Flow Cytometry, Software

Association of E2F2 expression and clinicopathological parameters in patients with CRC

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: Association of E2F2 expression and clinicopathological parameters in patients with CRC

Article Snippet: After blocked with 5% skim milk in tris-buffered saline tween (TBST) at room temperature for 2 h, the membranes were probed with the primary antibodies: mouse anti-human E2F2 monoclonal antibody (55 kDa; 1:1000) (sc-9967, Santa Cruz), rabbit anti-human GAPDH antibody (36 kDa; 1:1000) (sc-47724, Santa Cruz) at 4 °C overnight.

Techniques: Expressing

Expression patterns of E2F2 mRNA in CRC from TIMER, TCGA and GEO database. (A) The E2F2 expression in different cancer types from the TIMER database. (B) E2F2 mRNA expression was significantly downregulated in CRC tumor tissues compared to normal tissues from the TCGA-COADREAD data sets. (C) E2F2 mRNA expression was significantly decreased in paired CRC tumor tissues compared to adjacent normal tissues from the TCGA-COADREAD data sets. (D) E2F2 mRNA expression was significantly lower in colon adenocarcinoma than normal tissues from the GSE20916 dataset. (E) E2F2 mRNA expression was significantly reduced in early stage CRC tumor tissues compared to normal tissues from the GSE9348 dataset. (F-I) The mRNA expression level of E2F2 was analyzed using the TCGA-COADREAD data sets according to (F) T, (G) N, (H) M and (I) pathological stage. ns, no significant difference; * , p < 0.05; **, p < 0.01; ***, p < 0.001.

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: Expression patterns of E2F2 mRNA in CRC from TIMER, TCGA and GEO database. (A) The E2F2 expression in different cancer types from the TIMER database. (B) E2F2 mRNA expression was significantly downregulated in CRC tumor tissues compared to normal tissues from the TCGA-COADREAD data sets. (C) E2F2 mRNA expression was significantly decreased in paired CRC tumor tissues compared to adjacent normal tissues from the TCGA-COADREAD data sets. (D) E2F2 mRNA expression was significantly lower in colon adenocarcinoma than normal tissues from the GSE20916 dataset. (E) E2F2 mRNA expression was significantly reduced in early stage CRC tumor tissues compared to normal tissues from the GSE9348 dataset. (F-I) The mRNA expression level of E2F2 was analyzed using the TCGA-COADREAD data sets according to (F) T, (G) N, (H) M and (I) pathological stage. ns, no significant difference; * , p < 0.05; **, p < 0.01; ***, p < 0.001.

Techniques: Expressing

Logistic regression analysis of E2F2 expression associated with clinicopathological parameters in CRC

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: Logistic regression analysis of E2F2 expression associated with clinicopathological parameters in CRC

Techniques: Expressing

E2F2 expression level was reduced in clinical CRC tissues. (A-D) Representative images of E2F2 protein expression level in paraffin-embedded (A) normal mucosa and CRC tissues with (B) pathological stage I, (C) pathological stage II and (D) pathological stage III. Scales represent 100 micron. Representative images with original magnificent at 200×. (E) Quantifications of the average optical density (AOD) for E2F2 protein expression in normal mucosa and CRC tissues with pathological stage I-III. (F) Western blotting analysis and (G) quantitative real-time polymerase chain reaction analysis of five paired samples of CRC tissues and adjacent normal tissues from randomly selected CRC patients. * , p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: E2F2 expression level was reduced in clinical CRC tissues. (A-D) Representative images of E2F2 protein expression level in paraffin-embedded (A) normal mucosa and CRC tissues with (B) pathological stage I, (C) pathological stage II and (D) pathological stage III. Scales represent 100 micron. Representative images with original magnificent at 200×. (E) Quantifications of the average optical density (AOD) for E2F2 protein expression in normal mucosa and CRC tissues with pathological stage I-III. (F) Western blotting analysis and (G) quantitative real-time polymerase chain reaction analysis of five paired samples of CRC tissues and adjacent normal tissues from randomly selected CRC patients. * , p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

The diagnostic and prognostic value of E2F2 in CRC. (A-C) Kaplan-Meier survival curve analysis of OS, DFS and DSS showed that low E2F2 expression correlated to poor prognosis of CRC patients in a CRC cohort (GSE17537) from the PrognoScan database. (D-F) Survival curves showed OS, PFI and DSS rates of CRC patients with low or high E2F2 expression from the TCGA-COADREAD data sets. (G, H) ROC analysis illustrated that E2F2 expression accurately discriminated CRC tumor tissues from the normal tissues with an AUC of 0.865 (95% CI = 0.784-0.946) from GSE20916 data set and an AUC of 0.735 (95% CI = 0.672-0.798) from TCGA-COADREAD data sets. OS, overall survivial; DFS, disease free survival; DSS, disease specific survival; PFI, progress free interval; ROC: Receiver operating characteristic; AUC: area under the curve.

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: The diagnostic and prognostic value of E2F2 in CRC. (A-C) Kaplan-Meier survival curve analysis of OS, DFS and DSS showed that low E2F2 expression correlated to poor prognosis of CRC patients in a CRC cohort (GSE17537) from the PrognoScan database. (D-F) Survival curves showed OS, PFI and DSS rates of CRC patients with low or high E2F2 expression from the TCGA-COADREAD data sets. (G, H) ROC analysis illustrated that E2F2 expression accurately discriminated CRC tumor tissues from the normal tissues with an AUC of 0.865 (95% CI = 0.784-0.946) from GSE20916 data set and an AUC of 0.735 (95% CI = 0.672-0.798) from TCGA-COADREAD data sets. OS, overall survivial; DFS, disease free survival; DSS, disease specific survival; PFI, progress free interval; ROC: Receiver operating characteristic; AUC: area under the curve.

Techniques: Diagnostic Assay, Expressing

Univariate and multivariate analysis of clinicopathological factors that correlate with OS of CRC patients

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: Univariate and multivariate analysis of clinicopathological factors that correlate with OS of CRC patients

Techniques:

Go and KEGG enrichment analysis of genes related to E2F2 in CRC tissues in the TCGA-COADREAD data sets. (A-C) Go enrichment analysis showed the BP (biological processes), CC (cellular components), and MF (molecular function) of co-expressed genes with E2F2. (D) Significantly enriched KEGG terms obtained from KEGG enrichment analysis of co-expressed genes with E2F2.

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: Go and KEGG enrichment analysis of genes related to E2F2 in CRC tissues in the TCGA-COADREAD data sets. (A-C) Go enrichment analysis showed the BP (biological processes), CC (cellular components), and MF (molecular function) of co-expressed genes with E2F2. (D) Significantly enriched KEGG terms obtained from KEGG enrichment analysis of co-expressed genes with E2F2.

Techniques:

Enrichment plots from the gene set enrichment analysis (GSEA). (A) ATR pathway, (B) ATM signalling pathway, (C) mismatch repair, (D) base excision repair, (E) homologous recomibination, (F) Fanconi Anemia pathway, (G) multicancer invasiveness signature, (H) stem cell up, and (I) mammary stem cell up were significantly enriched in E2F2-related CRC. NES, normalized enrichment scores; FDR, false discovery rate.

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: Enrichment plots from the gene set enrichment analysis (GSEA). (A) ATR pathway, (B) ATM signalling pathway, (C) mismatch repair, (D) base excision repair, (E) homologous recomibination, (F) Fanconi Anemia pathway, (G) multicancer invasiveness signature, (H) stem cell up, and (I) mammary stem cell up were significantly enriched in E2F2-related CRC. NES, normalized enrichment scores; FDR, false discovery rate.

Techniques:

Association analysis of E2F2 gene expression and immune infiltration. (A) The association between E2F2 expression and 24 tumor-infiltrating lymphocytes. (B-G) The correlation of E2F2 expression with immune infiltration level of (B) Th2 cells, (C) aDC cells, (D) Th17 cells, (E) NK CD56dim cells, (F) T helper cells, (G) pDC cells.

Journal: Journal of Cancer

Article Title: Decreased E2F2 Expression Correlates with Poor Prognosis and Immune Infiltrates in Patients with Colorectal Cancer

doi: 10.7150/jca.61415

Figure Lengend Snippet: Association analysis of E2F2 gene expression and immune infiltration. (A) The association between E2F2 expression and 24 tumor-infiltrating lymphocytes. (B-G) The correlation of E2F2 expression with immune infiltration level of (B) Th2 cells, (C) aDC cells, (D) Th17 cells, (E) NK CD56dim cells, (F) T helper cells, (G) pDC cells.

Techniques: Gene Expression, Expressing

JMJD6 forms complexes with N-Myc and BRD4 to induce E2F2 and Myc expression. a Protein extracted from CHP134 cells was immunoprecipitated (IP) overnight with 2.5 μg of control IgG, anti-JMJD6 or anti-BRD4 antibody (Ab). The immunoprecipitated protein was immunoblotted with anti-JMJD6 or anti-BRD4 Ab. b , c DOX-inducible control shRNA, JMJD6 shRNA-1 or JMJD6 shRNA-2 CHP134, and SK-N-AS cells were treated with vehicle control or DOX for 48 h, followed by RT-PCR analysis of JMJD6 mRNA ( b ), or RT-PCR and immunoblot analysis of JMJD6, N-Myc, c-Myc, and E2F2 mRNA and protein ( c ). Error bars represent normalized standard errors from three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed unpaired Student’s t test). d Protein was extracted from CHP134 and SK-N-AS cells stably transfected with an empty vector or JMJD6 expression construct, followed by immunoblot analysis. e Protein extracted from CHP134 cells was subjected to IP overnight with 5 μg of control IgG or anti-N-Myc Ab, followed by immunoblot analysis with anti-JMJD6 or anti-N-Myc Ab. f Immobilized GST-N-Myc protein fragments were incubated with equal amounts of nuclear protein prepared from HEK-293T cells transiently transfected with the recombinant vector pCMV14-JMJD6_3 × Flag. Pulled-down complexes were probed with a monoclonal anti-Flag antibody, and Ponceau staining detected by ChemiDoc MP was used as loading controls. g DNA-protein complex was extracted from CHP134 cells for ChIP sequencing with anti-JMJD6 and anti-N-Myc antibodies. Numbers inside the Venn diagram indicated the numbers of peaks at super-enhancers, typical enhancers, and promoters, which were bound by JMJD6 or N-Myc, and the overlap between them. Source data are provided as a Source Data file

Journal: Nature Communications

Article Title: JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma

doi: 10.1038/s41467-019-11132-w

Figure Lengend Snippet: JMJD6 forms complexes with N-Myc and BRD4 to induce E2F2 and Myc expression. a Protein extracted from CHP134 cells was immunoprecipitated (IP) overnight with 2.5 μg of control IgG, anti-JMJD6 or anti-BRD4 antibody (Ab). The immunoprecipitated protein was immunoblotted with anti-JMJD6 or anti-BRD4 Ab. b , c DOX-inducible control shRNA, JMJD6 shRNA-1 or JMJD6 shRNA-2 CHP134, and SK-N-AS cells were treated with vehicle control or DOX for 48 h, followed by RT-PCR analysis of JMJD6 mRNA ( b ), or RT-PCR and immunoblot analysis of JMJD6, N-Myc, c-Myc, and E2F2 mRNA and protein ( c ). Error bars represent normalized standard errors from three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed unpaired Student’s t test). d Protein was extracted from CHP134 and SK-N-AS cells stably transfected with an empty vector or JMJD6 expression construct, followed by immunoblot analysis. e Protein extracted from CHP134 cells was subjected to IP overnight with 5 μg of control IgG or anti-N-Myc Ab, followed by immunoblot analysis with anti-JMJD6 or anti-N-Myc Ab. f Immobilized GST-N-Myc protein fragments were incubated with equal amounts of nuclear protein prepared from HEK-293T cells transiently transfected with the recombinant vector pCMV14-JMJD6_3 × Flag. Pulled-down complexes were probed with a monoclonal anti-Flag antibody, and Ponceau staining detected by ChemiDoc MP was used as loading controls. g DNA-protein complex was extracted from CHP134 cells for ChIP sequencing with anti-JMJD6 and anti-N-Myc antibodies. Numbers inside the Venn diagram indicated the numbers of peaks at super-enhancers, typical enhancers, and promoters, which were bound by JMJD6 or N-Myc, and the overlap between them. Source data are provided as a Source Data file

Article Snippet: The membranes were blocked of nonspecific antibody binding with 10% skim milk powder in phosphate-buffered saline, and probed with the following primary antibodies: mouse anti-E2F2 antibody (1:2000) (sc-633×), mouse anti-JMJD6 antibody (1:500) (sc-28348), mouse anti-N-Myc antibody (1:1000) (sc-53993), or rabbit anti-c-Myc antibody (1:500) (sc-764) (all from Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Control, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test, Stable Transfection, Transfection, Plasmid Preparation, Construct, Incubation, Recombinant, Staining, ChIP-sequencing

JMJD6 is important for E2F2 and Myc expression and tumor progression in vivo. a DOX-inducible JMJD6 shRNA-2 SK-N-AS cells were xenografted into nude mice. When tumors reached 0.05 cm 3 in volume, the mice were divided into two groups, and fed with food with or without 600 mg/kg of DOX. Tumor growth was monitored every other day, and the mice were culled when tumor reached 1 cm 3 . Error bars represent standard errors (** p < 0.01, two-tailed paired Student’s t test). b Survival curve analysis showed the probability of mouse overall survival. Two-sided log-rank test was used to generate the p- value. c , d Protein was extracted from the tumors from the mice, and subjected to immunoblot analysis of JMJD6, c-Myc, and E2F2 protein expression ( c ), followed by quantification against the house-keeping actin using Quantity One software ( d ). Error bars represent standard errors (** p < 0.01, *** p < 0.001, two-tailed unpaired Student’s t test). Source data are provided as a Source Data file

Journal: Nature Communications

Article Title: JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma

doi: 10.1038/s41467-019-11132-w

Figure Lengend Snippet: JMJD6 is important for E2F2 and Myc expression and tumor progression in vivo. a DOX-inducible JMJD6 shRNA-2 SK-N-AS cells were xenografted into nude mice. When tumors reached 0.05 cm 3 in volume, the mice were divided into two groups, and fed with food with or without 600 mg/kg of DOX. Tumor growth was monitored every other day, and the mice were culled when tumor reached 1 cm 3 . Error bars represent standard errors (** p < 0.01, two-tailed paired Student’s t test). b Survival curve analysis showed the probability of mouse overall survival. Two-sided log-rank test was used to generate the p- value. c , d Protein was extracted from the tumors from the mice, and subjected to immunoblot analysis of JMJD6, c-Myc, and E2F2 protein expression ( c ), followed by quantification against the house-keeping actin using Quantity One software ( d ). Error bars represent standard errors (** p < 0.01, *** p < 0.001, two-tailed unpaired Student’s t test). Source data are provided as a Source Data file

Techniques: Expressing, In Vivo, shRNA, Two Tailed Test, Western Blot, Software

THZ1 and panobinostat synergistically reduce JMJD6, E2F2, and Myc expression. a ChIP-Seq was performed with control IgG, mono-methyl H3K4 (H3K4me), trimethyl H3K4 (H3K4me3), or acetyl H3K27 (H3K27ac) antibodies in CHP134 cells. H3K4me, H3K4me3, and H3K27ac occupancy at the MXRA7 and JMJD6 gene loci was analyzed. The x- axis indicates genomic position and the y -axis depicts the ChIP-seq signal from each histone mark, in arbitrary units normalized against input control, and super enhancer regions were boxed in red. b ChIP-Seq profiling for H3K27ac occupancy at the MXRA7 and JMJD6 gene loci in SK-N-AS cells was analyzed, using the published Series GSE90683 dataset. c CHP134 and SK-N-AS cells were treated with vehicle control, 32 or 64 nM THZ1. RNA and protein were extracted from the cells 24 and 48 h later, respectively, and subjected to RT-PCR and immunoblot analysis of JMJD6, N-Myc, and c-Myc mRNA and protein expression. Error bars represent normalized standard errors from three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA). d CHP134 and SK-N-AS cells were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or combination of THZ1 and panobinostat for 48 h, followed by RT-PCR and immunoblot analyses of JMJD6, N-Myc, and c-Myc mRNA and protein expression. Error bars represent normalized standard errors from four independent experiments (* p < 0.05, ** p < 0.01, two-tailed unpaired Student’s t test). Source data are provided as a Source Data file

Journal: Nature Communications

Article Title: JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma

doi: 10.1038/s41467-019-11132-w

Figure Lengend Snippet: THZ1 and panobinostat synergistically reduce JMJD6, E2F2, and Myc expression. a ChIP-Seq was performed with control IgG, mono-methyl H3K4 (H3K4me), trimethyl H3K4 (H3K4me3), or acetyl H3K27 (H3K27ac) antibodies in CHP134 cells. H3K4me, H3K4me3, and H3K27ac occupancy at the MXRA7 and JMJD6 gene loci was analyzed. The x- axis indicates genomic position and the y -axis depicts the ChIP-seq signal from each histone mark, in arbitrary units normalized against input control, and super enhancer regions were boxed in red. b ChIP-Seq profiling for H3K27ac occupancy at the MXRA7 and JMJD6 gene loci in SK-N-AS cells was analyzed, using the published Series GSE90683 dataset. c CHP134 and SK-N-AS cells were treated with vehicle control, 32 or 64 nM THZ1. RNA and protein were extracted from the cells 24 and 48 h later, respectively, and subjected to RT-PCR and immunoblot analysis of JMJD6, N-Myc, and c-Myc mRNA and protein expression. Error bars represent normalized standard errors from three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA). d CHP134 and SK-N-AS cells were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or combination of THZ1 and panobinostat for 48 h, followed by RT-PCR and immunoblot analyses of JMJD6, N-Myc, and c-Myc mRNA and protein expression. Error bars represent normalized standard errors from four independent experiments (* p < 0.05, ** p < 0.01, two-tailed unpaired Student’s t test). Source data are provided as a Source Data file

Techniques: Expressing, ChIP-sequencing, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

THZ1 and panobinostat synergistically induce tumor regression by reducing JMJD6. a , b CHP134 and SK-N-AS cells were treated with vehicle control, THZ1, panobinostat, or their combination for 72 h, followed by Alamar blue assays ( a ). Error bars represent normalized standard errors from three independent experiments. Isobolograms showed the actual concentrations of THZ1 and panobinostat required to achieve inhibition of cell viability by 85% (IC 85 , solid line), as compared with additivity [dotted line, combination index (CI) = 1] ( b ). c CHP134 and SK-N-AS cells stably transfected with an empty vector or JMJD6 expression construct were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by Alamar blue assays. Error bars represent normalized standard errors from four independent experiments (* p < 0.05, *** p < 0.001, two-tailed unpaired Student’s t test). d CHP134 and SK-N-AS cells were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by staining with propidium iodide and flow cytometry analysis of the cell cycle. Error bars represent standard errors from four independent experiments (* p < 0.05, two-tailed unpaired Student’s t test). e Seventy-two nude mice were xenografted with SK-N-AS cells stably transfected with an empty vector or JMJD6 ORF expression construct. When tumors reached 0.05 mm 3 , the mice were divided into four subgroups of nice mice each and treated intraperitoneally with control solvent, THZ1 (10 mg/kg body weight/twice a day), panobinostat (7.5 mg/kg body weight/3 days), or THZ1 plus panobinostat for 21 days. Tumor mass was measured every other day. Error bars represent standard errors (* p < 0.05, *** p < 0.001, two-way ANOVA). f Protein was extracted from the tumor tissues, and subjected to immunoblot analysis of JMJD6 and E2F2 protein expression. Source data are provided as a Source Data file

Journal: Nature Communications

Article Title: JMJD6 is a tumorigenic factor and therapeutic target in neuroblastoma

doi: 10.1038/s41467-019-11132-w

Figure Lengend Snippet: THZ1 and panobinostat synergistically induce tumor regression by reducing JMJD6. a , b CHP134 and SK-N-AS cells were treated with vehicle control, THZ1, panobinostat, or their combination for 72 h, followed by Alamar blue assays ( a ). Error bars represent normalized standard errors from three independent experiments. Isobolograms showed the actual concentrations of THZ1 and panobinostat required to achieve inhibition of cell viability by 85% (IC 85 , solid line), as compared with additivity [dotted line, combination index (CI) = 1] ( b ). c CHP134 and SK-N-AS cells stably transfected with an empty vector or JMJD6 expression construct were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by Alamar blue assays. Error bars represent normalized standard errors from four independent experiments (* p < 0.05, *** p < 0.001, two-tailed unpaired Student’s t test). d CHP134 and SK-N-AS cells were treated with vehicle control, 32 nM THZ1, 10 nM panobinostat, or their combination for 72 h, followed by staining with propidium iodide and flow cytometry analysis of the cell cycle. Error bars represent standard errors from four independent experiments (* p < 0.05, two-tailed unpaired Student’s t test). e Seventy-two nude mice were xenografted with SK-N-AS cells stably transfected with an empty vector or JMJD6 ORF expression construct. When tumors reached 0.05 mm 3 , the mice were divided into four subgroups of nice mice each and treated intraperitoneally with control solvent, THZ1 (10 mg/kg body weight/twice a day), panobinostat (7.5 mg/kg body weight/3 days), or THZ1 plus panobinostat for 21 days. Tumor mass was measured every other day. Error bars represent standard errors (* p < 0.05, *** p < 0.001, two-way ANOVA). f Protein was extracted from the tumor tissues, and subjected to immunoblot analysis of JMJD6 and E2F2 protein expression. Source data are provided as a Source Data file

Techniques: Control, Inhibition, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Construct, Two Tailed Test, Staining, Flow Cytometry, Solvent, Western Blot

MMTV- Myc transgenic mice were interbred with E2F2 −/− mice and tumor latency was examined. Myc tumors developing in the absence of E2F2 had a significantly increased time to tumor onset ( A. p = 0.0057). Metastasis is rarely observed in MMTV- Myc mice with only 13% of tumor bearing mice having lung metastasis ( B. n = 2/15). Metastatic incidence is increased to 67% when Myc tumors develop in the E2F2 knockout background ( n = 6/9; p -value = 0.0361). Histology of a MMTV- Myc mouse lung showing the absence of lung metastasis at 4X C. compared with the metastases observed in the MMTV- Myc E2F2 null strain D. Increased magnification (20X) of these sections revealed secondary structure within the metastatic lesion E and F. To ensure that the metastatic effect of E2F2 loss was not a strain specific effect, MMTV- Myc WT21 mice were interbred with E2F2 −/− mice. Loss of E2F2 in the MMTV- Myc WT21 background resulted in decreased latency G. and trend towards increased percentage of metastasis bearing mice H. Transplantation of MMTV- Myc WT21 E2F2 −/− tumors into MMTV- Myc WT21 or MMTV- Myc WT21 E2F2 −/− backgrounds produced striking metastases, suggesting that loss of E2F2 affected metastasis in a cell autonomous manner I.

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: MMTV- Myc transgenic mice were interbred with E2F2 −/− mice and tumor latency was examined. Myc tumors developing in the absence of E2F2 had a significantly increased time to tumor onset ( A. p = 0.0057). Metastasis is rarely observed in MMTV- Myc mice with only 13% of tumor bearing mice having lung metastasis ( B. n = 2/15). Metastatic incidence is increased to 67% when Myc tumors develop in the E2F2 knockout background ( n = 6/9; p -value = 0.0361). Histology of a MMTV- Myc mouse lung showing the absence of lung metastasis at 4X C. compared with the metastases observed in the MMTV- Myc E2F2 null strain D. Increased magnification (20X) of these sections revealed secondary structure within the metastatic lesion E and F. To ensure that the metastatic effect of E2F2 loss was not a strain specific effect, MMTV- Myc WT21 mice were interbred with E2F2 −/− mice. Loss of E2F2 in the MMTV- Myc WT21 background resulted in decreased latency G. and trend towards increased percentage of metastasis bearing mice H. Transplantation of MMTV- Myc WT21 E2F2 −/− tumors into MMTV- Myc WT21 or MMTV- Myc WT21 E2F2 −/− backgrounds produced striking metastases, suggesting that loss of E2F2 affected metastasis in a cell autonomous manner I.

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Transgenic Assay, Knock-Out, Transplantation Assay, Produced

Gene expression analysis of MMTV- Myc tumors in E2F the WT, E2F1 −/− , E2F2 −/− and E2F3 +/− backgrounds and 6 sample of lung metastasis were analyzed by microarray. Unsupervised hierarchical clustering revealed that samples were clustered based on their histological type rather than their genotype with lung metastasis samples being clustered together A. The clustering of the metastatic samples in one group, closely related to the papillary subtype was noted. We examined the indicated sub-clusters of genes for predicted transcription factor binding demonstrating that genes upregulated in metastasis, represented in cluster D, were enriched for E2F binding motifs B. Comparison of EMT and lung metastasis samples by GSEA demonstrated enrichment of genes regulated by SMAD2, SMAD3 , and SMAD4 was elevated in EMT relative to lung metastases ( C. p = 0.03). Enrichment of genes up-regulated in invasive ovarian epithelial cancer was noted in the metastasis samples ( D. p = 0.016).

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: Gene expression analysis of MMTV- Myc tumors in E2F the WT, E2F1 −/− , E2F2 −/− and E2F3 +/− backgrounds and 6 sample of lung metastasis were analyzed by microarray. Unsupervised hierarchical clustering revealed that samples were clustered based on their histological type rather than their genotype with lung metastasis samples being clustered together A. The clustering of the metastatic samples in one group, closely related to the papillary subtype was noted. We examined the indicated sub-clusters of genes for predicted transcription factor binding demonstrating that genes upregulated in metastasis, represented in cluster D, were enriched for E2F binding motifs B. Comparison of EMT and lung metastasis samples by GSEA demonstrated enrichment of genes regulated by SMAD2, SMAD3 , and SMAD4 was elevated in EMT relative to lung metastases ( C. p = 0.03). Enrichment of genes up-regulated in invasive ovarian epithelial cancer was noted in the metastasis samples ( D. p = 0.016).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Gene Expression, Microarray, Binding Assay, Comparison

To examine the regulatory mechanisms involved in E2F2 -mediated human breast cancer metastasis, we established a pipeline for analysis. Genes that were differentially expressed by MMTV- Myc , MMTV- Myc x E2F2 −/− and lung metastasis samples A–C. were identified. Mouse gene expression data for significantly expressed genes was clustered together with human datasets, revealing 451 candidate genes that are homologous to human genes. We ranked these potential target genes based on their correlation with human distant metastasis free survival, their cox-hazard ratio, and the existence of E2F motifs proximal to the transcriptional start site. Comparison between MMTV- Myc tumors and lung metastases revealed elevated PTPRD expression in the lung metastases samples D. Comparison of MMTV- Myc WT21 non-metastatic tumors and MMTV- Myc WT21 E2F2 −/− metastatic tumors passaged in their respective genotype revealed increased PTPRD expression in the MMTV- Myc WT21 E2F2 −/− tumors ( E. D = donor, R = recipient). Elevated levels of PTPRD were found to be correlated with human distant metastasis free survival ( F. p = 0.0153) and is associated with basal breast cancer subtype ( G. p = 0.0085).

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: To examine the regulatory mechanisms involved in E2F2 -mediated human breast cancer metastasis, we established a pipeline for analysis. Genes that were differentially expressed by MMTV- Myc , MMTV- Myc x E2F2 −/− and lung metastasis samples A–C. were identified. Mouse gene expression data for significantly expressed genes was clustered together with human datasets, revealing 451 candidate genes that are homologous to human genes. We ranked these potential target genes based on their correlation with human distant metastasis free survival, their cox-hazard ratio, and the existence of E2F motifs proximal to the transcriptional start site. Comparison between MMTV- Myc tumors and lung metastases revealed elevated PTPRD expression in the lung metastases samples D. Comparison of MMTV- Myc WT21 non-metastatic tumors and MMTV- Myc WT21 E2F2 −/− metastatic tumors passaged in their respective genotype revealed increased PTPRD expression in the MMTV- Myc WT21 E2F2 −/− tumors ( E. D = donor, R = recipient). Elevated levels of PTPRD were found to be correlated with human distant metastasis free survival ( F. p = 0.0153) and is associated with basal breast cancer subtype ( G. p = 0.0085).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Gene Expression, Comparison, Expressing

E2F2 knockdown in human breast cancer was achieved by transfection of MDA-MB-231 cells with sh E2F2 . Efficacy of E2F2 knockdown was assayed by western blotting A. with untransfected MDA-MB-231 (Con.), MDA-MB-231 transfected with shScramble (Scr.), sh E2F2 construct #3 (C3), sh E2F2 construct #4 (C4). Migration of MDA-MB-231 control cells B. and with E2F2 knockdown C. in transwell migration assays revealed that the percentage of cells that migrated across the membrane increased when the level of E2F2 was decreased ( D. p < 0.0001). In colonization assays with and without the knockdown, lesions were found in the lungs of mice injected with MDA-MB-231 E. and greatly increased with transfection of sh E2F2 F. Quantification of the numbers of metastatic lesions revealed an increased number of metastatic lesions in mice injected with MDA-MB-231 transfected with sh E2F2 ( G. p = 0.0184).

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: E2F2 knockdown in human breast cancer was achieved by transfection of MDA-MB-231 cells with sh E2F2 . Efficacy of E2F2 knockdown was assayed by western blotting A. with untransfected MDA-MB-231 (Con.), MDA-MB-231 transfected with shScramble (Scr.), sh E2F2 construct #3 (C3), sh E2F2 construct #4 (C4). Migration of MDA-MB-231 control cells B. and with E2F2 knockdown C. in transwell migration assays revealed that the percentage of cells that migrated across the membrane increased when the level of E2F2 was decreased ( D. p < 0.0001). In colonization assays with and without the knockdown, lesions were found in the lungs of mice injected with MDA-MB-231 E. and greatly increased with transfection of sh E2F2 F. Quantification of the numbers of metastatic lesions revealed an increased number of metastatic lesions in mice injected with MDA-MB-231 transfected with sh E2F2 ( G. p = 0.0184).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques: Knockdown, Transfection, Western Blot, Construct, Migration, Control, Membrane, Injection

We examined a protein-protein interaction network and found that E2F2 and PTPRD were connected through MYC and STAT3 A. Unsupervised clustering of human breast tumor datasets and mouse tumor dataset revealed a cluster in which lung metastasis samples and human tumors were clustered B. Stratification of human Distant Metastasis Free Survival data by E2F2 pathway probability values showed differential effect of E2F2 loss in human tumors C. p -value < 0.0001).

Journal: Oncotarget

Article Title: Increased metastasis with loss of E2F2 in Myc -driven tumors

doi:

Figure Lengend Snippet: We examined a protein-protein interaction network and found that E2F2 and PTPRD were connected through MYC and STAT3 A. Unsupervised clustering of human breast tumor datasets and mouse tumor dataset revealed a cluster in which lung metastasis samples and human tumors were clustered B. Stratification of human Distant Metastasis Free Survival data by E2F2 pathway probability values showed differential effect of E2F2 loss in human tumors C. p -value < 0.0001).

Article Snippet: Primary antibodies for immunoblotting were rabbit anti- E2F2 (clone E-19 Santa Cruz, Dallas, TX, 1:300) or rabbit anti- PTPRD (Abcam, Cambridge, MA, 1:100).

Techniques:

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