Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 1. Association between gene signature and PFS of NMIBC in the Korean cohort (n ¼ 102). A, a predominant signaling pathway consisting of E2F1, EZH2, and SUZ12 associated with disease progression of NMIBC. Up- and downregulated genes in the EH group are indicated in red and green, respectively. The intensity of color is indicative of the degree of over- or underexpression. Each line and arrow represents functional and physical interactions between the genes and the direction of regulation reported in the literature. B, two-group boxplot comparing expression levels of E2F1, EZH2, and SUZ12 in EH (a high E2F1 cluster) and EL (a low E2F1 cluster) patients. Each P value was obtained by two-sample t test between EH and EL. C, survival estimation, NMIBC progression, MIBC OS, with a signature composed of 3 genes, E2F1, EZH2, and SUZ12.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Biomarker Discovery, Functional Assay, Expressing

Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 2. Alternative expression of E2F1, EZH2, and SUZ12 regulates invasion and migration in bladder cancer. A and B, effects of overexpression of E2F1, EZH2, and SUZ12 genes in UC9 cells. A, cells overexpressing pE2F1, pEZH2, or pSUZ12 compared with cells with control vector (pcDNA6). Upregulation of mRNA and protein expression were detected by RT-PCR and Western blotting. B, cell overexpressing E2F1, EZH2, and SUZ12 showed increased cell migration and invasion. C and D, effects of knockdown (KD) of E2F1, EZH2, and SUZ12 genes in EJ cells. C, downregulation of mRNA and protein expression was detected in cells with siE2F1, siEZH2, or siSUZ12 compared with cells with control (scRNA). Downregulated E2F1, EZH2, and SUZ12 cells showed reduced invasion and migration. Data are presented as mean SD for 3 independent experiments (original magnification, 200). , P < 0.001.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Expressing, Migration, Over Expression, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown

Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 3. Effect of alternative expression of E2F1, EZH2, and SUZ12 on cell proliferation and tumorigenesis. A, UC9 cells overexpressing E2F1, EZH2, or SUZ12 showed significantly higher cell proliferation and cell viability than that of empty vector–transfected cells (pcDNA6; left). The siE2F1, siEZH2, or siSUZ12-KD EJ cells showed decreased cell proliferation and cell viability (right). B, tumor volume from xenografted nude mice with control shRNA, shE2F1, shEZH2, or shSUZ12 cell lines. Injection of shE2F1, shEZH2, or shSUZ12 cell lines showed a significant decrease in tumor growth compared with control-treated groups. Volumes of tumors dissected from the sacrificed mice (, P < 0.01). The decreased RNA and protein levels from the resected tumors are represented in right. C, tumor volume from xenograftednude mice with transfected UC5 cells with control pCDNA, pE2F1, pEZH2, or pSUZ12 overexpression vector. Injection of transfected pE2F1, pEZH2, or pSUZ12 cell lines showed a significant increase in tumor growth compared with control pCDNA groups. Volumes of tumors dissected from the sacrificed mice (, P < 0.01). The increased RNA and protein levels from the resected tumors are represented in right.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Expressing, Plasmid Preparation, Transfection, Control, shRNA, Injection, Over Expression

Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 5. E2F1 controls expression of EZH2 and SUZ12 in bladder cancer cells. A, E2F1 KD decreases the expression of EZH2 and SUZ12 in bladder cancer cells. Left, UC9 cells show upregulation of EZH2 and SUZ12 by E2F1 overexpression. Right, EJ cells show underexpressed EZH2 and SUZ12 expression with E2F1-targeted siRNA (siE2F1) compared with scrambled control (scRNA). B, E2F1 KD (shE2F1) leads to downregulation of EZH2 and SUZ12 when compared with controls (shCon). Downregulation was suppressed by pcDNA6-EZH2 or -SUZ12 overexpression vectors, but not by pcDNA empty vector. C, representative images of entire invaded and stained chambers are shown. Decreased invasion of EJ cells with E2F1-targeted shRNA (shE2F1#1, #2) compared with scrambled shRNA (NTS). Reduced invasive ability was effectively suppressed by EZH2 (þpEZH2) or SUZ12 (þpSUZ12) overexpression but not by pcDNA empty vector. D, results shown on the graph represent means SD of 3 independent experiments. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Expressing, Over Expression, Control, Plasmid Preparation, Staining, shRNA

Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 1. Association between gene signature and PFS of NMIBC in the Korean cohort (n ¼ 102). A, a predominant signaling pathway consisting of E2F1, EZH2, and SUZ12 associated with disease progression of NMIBC. Up- and downregulated genes in the EH group are indicated in red and green, respectively. The intensity of color is indicative of the degree of over- or underexpression. Each line and arrow represents functional and physical interactions between the genes and the direction of regulation reported in the literature. B, two-group boxplot comparing expression levels of E2F1, EZH2, and SUZ12 in EH (a high E2F1 cluster) and EL (a low E2F1 cluster) patients. Each P value was obtained by two-sample t test between EH and EL. C, survival estimation, NMIBC progression, MIBC OS, with a signature composed of 3 genes, E2F1, EZH2, and SUZ12.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Biomarker Discovery, Functional Assay, Expressing

Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 2. Alternative expression of E2F1, EZH2, and SUZ12 regulates invasion and migration in bladder cancer. A and B, effects of overexpression of E2F1, EZH2, and SUZ12 genes in UC9 cells. A, cells overexpressing pE2F1, pEZH2, or pSUZ12 compared with cells with control vector (pcDNA6). Upregulation of mRNA and protein expression were detected by RT-PCR and Western blotting. B, cell overexpressing E2F1, EZH2, and SUZ12 showed increased cell migration and invasion. C and D, effects of knockdown (KD) of E2F1, EZH2, and SUZ12 genes in EJ cells. C, downregulation of mRNA and protein expression was detected in cells with siE2F1, siEZH2, or siSUZ12 compared with cells with control (scRNA). Downregulated E2F1, EZH2, and SUZ12 cells showed reduced invasion and migration. Data are presented as mean SD for 3 independent experiments (original magnification, 200). , P < 0.001.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Expressing, Migration, Over Expression, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown

Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 3. Effect of alternative expression of E2F1, EZH2, and SUZ12 on cell proliferation and tumorigenesis. A, UC9 cells overexpressing E2F1, EZH2, or SUZ12 showed significantly higher cell proliferation and cell viability than that of empty vector–transfected cells (pcDNA6; left). The siE2F1, siEZH2, or siSUZ12-KD EJ cells showed decreased cell proliferation and cell viability (right). B, tumor volume from xenografted nude mice with control shRNA, shE2F1, shEZH2, or shSUZ12 cell lines. Injection of shE2F1, shEZH2, or shSUZ12 cell lines showed a significant decrease in tumor growth compared with control-treated groups. Volumes of tumors dissected from the sacrificed mice (, P < 0.01). The decreased RNA and protein levels from the resected tumors are represented in right. C, tumor volume from xenograftednude mice with transfected UC5 cells with control pCDNA, pE2F1, pEZH2, or pSUZ12 overexpression vector. Injection of transfected pE2F1, pEZH2, or pSUZ12 cell lines showed a significant increase in tumor growth compared with control pCDNA groups. Volumes of tumors dissected from the sacrificed mice (, P < 0.01). The increased RNA and protein levels from the resected tumors are represented in right.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Expressing, Plasmid Preparation, Transfection, Control, shRNA, Injection, Over Expression

Journal: Clinical Cancer Research

Article Title: Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer

doi: 10.1158/1078-0432.ccr-14-2680

Figure Lengend Snippet: Figure 5. E2F1 controls expression of EZH2 and SUZ12 in bladder cancer cells. A, E2F1 KD decreases the expression of EZH2 and SUZ12 in bladder cancer cells. Left, UC9 cells show upregulation of EZH2 and SUZ12 by E2F1 overexpression. Right, EJ cells show underexpressed EZH2 and SUZ12 expression with E2F1-targeted siRNA (siE2F1) compared with scrambled control (scRNA). B, E2F1 KD (shE2F1) leads to downregulation of EZH2 and SUZ12 when compared with controls (shCon). Downregulation was suppressed by pcDNA6-EZH2 or -SUZ12 overexpression vectors, but not by pcDNA empty vector. C, representative images of entire invaded and stained chambers are shown. Decreased invasion of EJ cells with E2F1-targeted shRNA (shE2F1#1, #2) compared with scrambled shRNA (NTS). Reduced invasive ability was effectively suppressed by EZH2 (þpEZH2) or SUZ12 (þpSUZ12) overexpression but not by pcDNA empty vector. D, results shown on the graph represent means SD of 3 independent experiments. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: The antibodies used in immunoblotting were against E2F1 (A300-766A, Bethyl Laboratories, Montgomery, TX) EZH2 (4905, Cell Signaling Technology Inc., Beverly, MA) SUZ12 (A302-407A, Bethyl Laboratories), and β- actin (4967, Cell Signaling Technology Inc.).

Techniques: Expressing, Over Expression, Control, Plasmid Preparation, Staining, shRNA

Journal: Scientific Reports

Article Title: The nucleoplasmic interactions among Lamin A/C-pRB-LAP2α-E2F1 are modulated by dexamethasone

doi: 10.1038/s41598-021-89608-3

Figure Lengend Snippet: Dex promoted the phosphorylation of S22 and S404 in Lamin A/C. Phosphorylations can participate in A-type lamin A type solubilization. ( A ) Typical western blot by using anti phospho S22 Lamin A/C antibody. Only in AT 648 hT cells was the phosphorylation increment statistically significant (Wilcoxon test p < 0.05, n = 7). ( B ) The typical western blot using the anti phospho S404 Lamin A/C antibody. AT 648 hT cells showed a statistically significant phosphorylation increment (Wilcoxon test p < 0.05, n = 8). Original images in the series of figures labeled S6O in supplementary information.

Article Snippet: The antibodies listed below were purchased from commercial sources: anti Lamin A/C (Cell Signaling Technology CST #4777 and Diatheva #ANT0072), FITC-conjugated anti Lamin A/C (CST #8617), anti LAP2α (BETHYL, A304-839A-M), anti phospho S22 Lamin A/C (CST #13448), anti phospho S404 Lamin A/C was kindly provided by Prof. Marmiroli , anti pRB (CST #9309, BETHYL #A302-433A-T), anti E2F1 (Santa Cruz Biotechnology #sc-251, BETHYL #A300-766A-M).

Techniques: Phospho-proteomics, Western Blot, Labeling

Journal: Science Advances

Article Title: Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control

doi: 10.1126/sciadv.aaw4640

Figure Lengend Snippet: ( A ) U2OS cells were treated with 5 μM PRMT5 inhibitor for 72 hours, where indicated, before ChIP analysis with anti-SUMO2/3–specific or control antibodies. Immunoprecipitated chromatin was analyzed using primers specific for the E2F site in the p73 promoter (i). An RT-PCR was also performed to monitor the levels of p73 transcripts in the cell (ii). An immunoblot for H4R3me2s is included to demonstrate the activity of the PRMT5 inhibitor (iii). n = 3. See also fig. S4 (F and G). ( B ) As described above, although cells were treated with the PRMT5 inhibitor for 24 or 48 hours as indicated. ChIP analysis was performed with anti-HP1α–specific or control antibodies (i). An immunoblot for H4R3me2s is included to demonstrate the activity of the PRMT5 inhibitor (ii). n = 2. ( C ) U2OS cells were transfected with SENP7 siRNA or nontargeting siRNA (siNT) for 96 hours as indicated. Cells were then prepared for ChIP analysis as described above (i). An immunoblot is included to demonstrate input protein levels (ii). n = 4. ( D ) ChIP analysis as described above, although U2OS cells were transfected with siRNA targeting E2F1, SENP7, or a combination of the two (siE2F1 + siSENP7). n = 3. ( E ) U2OS cells were transfected with siRNA targeting SENP7 or nontargeting siRNA for 96 hours, as indicated. Cells were subsequently transfected for 48 hours with an empty vector or a plasmid expressing Flag-tagged SENP7 V5. Cells were then prepared for ChIP analysis as described above (i). An immunoblot is included to demonstrate input protein levels (ii). n = 3. ( F ) U2OS cells were transfected with p73–luciferase (luc) or CDC6-luciferase reporter plasmids for 48 hours, along with empty vector (vec) or Flag-tagged SENP7 V5. Reporter activity was measured, and immunoblots were performed to monitor input protein levels. n = 2. ( G ) Model diagram where PRMT5-mediated methylation of chromatin-associated E2F1 mediates its interaction with p100/TSN, which permits the E2F1 complex to associate with a subset of RNAs, some being derived from E2F-target genes. By regulating the activity of the splicing machinery, it is proposed that the E2F1-p100/TSN complex can influence the alternative splicing of these RNAs. In the absence of E2F1 methylation (either under conditions of PRMT5 inhibitor treatment or in cells expressing E2F1-meR point mutants), a p100/TSN-dependent interaction with the splicing machinery is lost, and changes to alternative splicing of a subset of RNAs result.

Article Snippet: The following antibodies were used in immunoblots: p100/TSN (A302-883A, Bethyl Laboratories; RRID: AB_10631268), E2F1 (C20, Santa Cruz Biotechnology; RRID: AB_631394), E2F1 (A300-766A, Bethyl Laboratories; RRID: AB_2096774), HA (16B12, Covance; RRID: AB_10063630), FLAG (M2, Sigma-Aldrich; RRID: AB_262044), β-actin (AC-74, Sigma-Aldrich; RRID: AB_476697), H4R3me2s (ab5823, Abcam; RRID: AB_10562795), histone H4 (ab10158, Abcam; RRID: AB_296888), and SENP7 (donated by R. Hay, University of Dundee, UK).

Techniques: Control, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Methylation, Derivative Assay, Alternative Splicing