Journal: Cell death and differentiation

Article Title: E2F1-regulated miR-30b suppresses Cyclophilin D and protects heart from ischemia/reperfusion injury and necrotic cell death.

doi: 10.1038/cdd.2014.165

Figure Lengend Snippet: Figure 6 E2F1 promotes necrosis through miR-30b and CypD. (a) E2F1 levels are increased in cardiomyocytes exposed to H2O2. Cardiomyocytes were exposed to H2O2. Cells were harvested at the indicated times for the analysis of E2F1 levels by immunoblot. (b) Knockdown of E2F1 reduces necrotic cell death induced by H2O2. Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc. Twenty-four hours after infection, cells were treated with H2O2. PI exclusion was analyzed. *Po0.05 versus H2O2 alone. (c) The levels of E2F1 are increased in myocardial I/R. Mice were induced to undergo cardiac I/R at the indicated times as described in Materials and Methods. E2F1 levels were analyzed by immunoblot. (d and e) E2F1 knockout mice attenuates myocyte necrosis and myocardial infarction upon I/R. WTand E2F1 knockout mice were subjected to I/R as described in Materials and Methods. Myocyte necrosis (d) and myocardial infarction (e) were analyzed. *Po0.05 versus WT+I/R. (f and g) CypD TP attenuates the inhibitory effect of E2F1 knockdown on CypD expression and necrotic responses induced by H2O2. Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc, transfected with the TP (CypD-TP miR-30b) or the control (CypD-TP control) and then exposed to H2O2. CypD expression (f) was analyzed by immunoblot. Necrosis was assessed by PI exclusion assay (g). *Po0.05. GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: Mouse E2F1 cDNA was from Origene (Rockville, MD, USA).

Techniques: Western Blot, Knockdown, Infection, Knock-Out, Expressing, Transfection, Control, Exclusion Assay

Journal: eBioMedicine

Article Title: YAP1 activation is associated with the progression and response to immunotherapy of non-muscle invasive bladder cancer

doi: 10.1016/j.ebiom.2022.104092

Figure Lengend Snippet: YAP1 knockdown inhibits the proliferation of bladder cancer cells in vitro. The expression of YAP1 subgroup target genes was partially restored by YAP1 expression. (a) UC3 cells were infected with lentivirus expressing shYAP1 or scrambled shRNA (scRNA). YAP1 mRNA and protein levels were detected using qRT–PCR and Western blotting, respectively. (b) Cell viability was analysed at 0, 24, and 48 h with the MTT assay to determine the proliferation of the UC3 cell line. The data are presented as the means ± standard deviations of three independent experiments. (c) A clonogenic assay was performed with control and YAP1 knockdown UC3 cells. Cells were stained with a crystal violet solution, and colonies were counted in three independent experiments. Invasion and migration assays of UC3 cells transfected with shNTS and shYAP1 for 24 h were performed using Boyden chamber assays. Data are presented as the means ± standard deviations of three experiments. (d) The YAP1 activation 1 (YA1) genes FOXM1, E2F1, CCNA2, AURKA, BIRC5, LMNB1, and TEAD2 were validated at the mRNA level after YAP1 knockdown in the UC3 cell line. The mRNA levels of CTGF, CYR61, and ZEB1 included in YAP1 activation 2 (YA2) group were verified using qRT–PCR. The YAP1 inactivation (YI) genes GATA2, SMAD3, and SMAD6 were validated in YAP1 knockdown cells using qRT–PCR. The data are presented as the means ± standard deviations of three independent experiments. (e) Levels of the YAP1, CTGF, CYR61, E2F1, and FOXM1 proteins were detected using Western blotting. (f) In qRT–PCR experiments, the levels of YA1 and YA2 subgroup genes were detected following YAP1 overexpression, and the levels of YA1 and YA2 subgroup genes were restored by YAP1 overexpression. This experiment was performed three times. ns , not significant; *, p < 0·05; **, p < 0·01; and ***, p < 0·001.

Article Snippet: Primary antibodies against GAPDH (Cat# 2118, RRID: AB_561053, Cell Signaling Technology, MA, USA), YAP1 (Cat# 4912, RRID: AB_2218911, Cell Signaling Technology, MA, USA), p-YAP1 (S127) (Cat# 4911, RRID: AB_2218913, Cell Signaling Technology, MA, USA), CYR61 (Cat# sc-374129, RRID: AB_10947399, Santa Cruz Biotechnology, CA, USA), CCNA2 (Cat# NBP1-31330, RRID: AB_10003781, Novus Biologicals, CO, USA), E2F1 (Cat# A300-766A, RRID: AB_2096774, Bethyl Laboratories, TX, USA), and FOXM1 (Cat# A301-533A, RRID: AB_999586, Bethyl Laboratories, TX, USA) were used.

Techniques: Knockdown, In Vitro, Expressing, Infection, shRNA, Quantitative RT-PCR, Western Blot, MTT Assay, Clonogenic Assay, Control, Staining, Migration, Transfection, Activation Assay, Over Expression

Journal: Advanced Science

Article Title: Novel Evolutionarily Conserved Oncogene COA4 is Driven by KRAS Mutant and Promotes Cancer Metastasis Through Dual Mitochondrial Metabolism‐Dependent and ‐Independent Mechanisms

doi: 10.1002/advs.202507533

Figure Lengend Snippet: E2F1 directly binds to COA4 promoter and promotes its expression under KRAS mutation. A,B) Western blot (A) and RT‐qPCR (B) analyses of COA4 expression in A549 cells following E2F1 overexpression or knockdown. ( n = 3; ** p < 0.01; **** p < 0.0001) C) Luciferase reporter assay confirming that E2F1 directly regulates COA4 transcription in 293T and A549 cells. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) D) Luciferase reporter assay demonstrating that KRAS enhances E2F1‐mediated transcriptional activation of the COA4 promoter in 293T cells. ( n = 3; **** p < 0.0001) E) ChIP‐qPCR analysis identifying the specific E2F1 binding region within the COA4 promoter in A549 cells. ( n = 3; *** p < 0.001) F) AlphaFold 3 molecular docking of the COA4 promoter with E2F1, supporting their direct interaction. G) Chromatin immunoprecipitation followed by qPCR (ChIP‐qPCR) was performed to evaluate E2F1 binding to the COA4 promoter in normal tissues and KRAS‐mutant tumor tissues. Data are presented as mean ± SD ( n = 6, *** p < 0.001) H) Spearman's rank correlation analysis of COA4 and E2F1 expression in LUAD using the GEPIA2 database. I. IHC analysis of E2F1 expression in LUAD tumor versus normal tissues ( n = 12 per group; **** p < 0.0001) J) ATP quantification in E2F1 ‐overexpressing A549 cells following COA4 knockdown.( n = 3; ** p < 0.01) K) Complex IV enzyme activity in E2F1 ‐overexpressing A549 cells after COA4 knockdown. ( n = 3; ** p < 0.01) L,M) Western blot (L) and RT‐qPCR (M) analyses of COA4 expression in KRAS G12C ‐overexpressing A549 cells following E2F1 knockdown. ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) N) Western blot analysis of COA4 expression in A549 cells treated with the PI3K agonist 740Y‐P(15 µM) for 48 h (left), and in 740Y‐P‐treated A549 cells following E2F1 knockdown (right).( n = 3) O) Western blot analysis of COA4 expression in A549 and H23 cells treated with the PI3K inhibitor LY294002 following E2F1 overexpression. ( n = 3) P) RT‐qPCR analysis of COA4 mRNA levels in A549 cells treated with LY294002 following E2F1 overexpression (left), and in A549 cells treated with 740Y‐P following E2F1 knockdown (right). ( n = 3; ** p < 0.01; *** p < 0.001; **** p < 0.0001) Q) Transwell assays assessing migration of COA4 ‐overexpressing A549 and H23 cells following E2F1 knockdown (top), and of COA4‐knockdown cells following E2F1 overexpression (bottom). Scale bar: 275 µm (n = 3; ** p < 0.01; **** p < 0.0001) R) Transwell assays analyzing migration of A549 and H23 cells treated with 740Y‐P following E2F1 knockdown (left), and with LY294002 following E2F1 overexpression (right). Scale bar: 275 µm ( n = 3) S) Transwell assays evaluating migration and invasion in A549 and H23 cells following KRAS G12D overexpression, E2F1 knockdown, and COA4 overexpression. ( n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001) T) Analysis of E2F1 expression in LUAD tissues: left, comparing KRAS wild‐type versus KRAS mutant samples using TCGA data; right, comparing LUAD tissues with high versus low KRAS expression using combined datasets ( GSE30219 , GSE37745 , GSE118370 , and GSE140797 ). U) Survival analysis based on COA4 and E2F1 expression levels using the GSE30219 database. V) Combined analysis of COA4 and E2F1 expression with survival status and overall survival time, performed using the linkET package on the GSE30219 dataset. The data are given as mean ± SD and compared by Student's t test (A–E, G, I–T), Spearman's rank correlation analysis (H). The Log‐rank (Mantel–Cox) test was used for survival analyses (U,V). Significance levels are indicated as follows: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Primary antibodies: ACTIN (1:5000, Proteintech, 81115‐1‐RR); Coa4 (1:2500, Invitrogen, PA5‐59076); CDC42 (1:1000, Proteintech, 10155‐1‐AP); E2F1 (1:1000, Proteintech, 12171‐1‐AP); MTCO1 (1:500, ABclonal, A17889); MTCO2 (1:3000, Proteintech, 55070‐1‐AP); MTCO3 (1:2000, Proteintech, 55082‐1‐AP); KRAS (1:10000, Proteintech, 12063‐1‐AP); FAK (1:5000, ABclonal, A11131); p‐FAK‐Y397 (1:300, ABclonal, AP0302).

Techniques: Expressing, Mutagenesis, Western Blot, Quantitative RT-PCR, Over Expression, Knockdown, Luciferase, Reporter Assay, Activation Assay, ChIP-qPCR, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Migration

Journal: The World Journal of Men's Health

Article Title: Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2

doi: 10.5534/wjmh.220073

Figure Lengend Snippet: Expressions of EZH2 and E2F1 in DU 145 cells. WB assay shows decreases of EZH2 and E2F1 expressions after docetaxel treatment (A). Using knockdown of E2F1, the expressions of E2F1 and EZH2 are decreased (B), and TRAIL (100 ng/mL) treatment causes significant cell death in siE2F-1 DU 145 cells ( *** p<0.001) (C). DR4: death receptor 4, DR5: death receptor 5, EZH2: enhancer of zeste homolog 2, ns: not significant, TRAIL: tumor necrosis factor-related apoptosis-inducing ligand, WB: western blot.

Article Snippet: Small interfering RNA (siRNA) against human E2F1 (siE2F1) (sc-29297) and control siRNA (scRNA) (sc-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Western Blot

Journal: Cell death discovery

Article Title: p21 Regulates Wnt-Notch balance via DREAM/MMB/Rb-E2F1 and maintains intestinal stem cell homeostasis.

doi: 10.1038/s41420-024-02192-z

Figure Lengend Snippet: Fig. 4 The activated E2F1 up-regulated the transcription of Notch1, resulted in the activation of Notch pathway, and inhibition of Wnt pathway in p21G2TKO MEFs. A Co-IP with anti-E2F1 antibody indicate that E2F1 binding Rb reduced in p21G2TKO cells. B ChIP assay with anti-E2F1 antibody reveals that E2F1 binds Notch1 promoter with higher efficiency in p21G2TKO cells, suggesting the released E2F1 activity promoted Notch1 transcription. N = 3, **, p < 0.01; ***, p < 0.001. C Co-IP with anti-β-Catenin antibody indicated more Notch1 and Rbpj proteins are bound to β-Catenin in p21G2TKO cells, suggesting that the up-regulation of Notch1 results in its enhanced binding to β-Catenin. D ChIP assay with anti-β-Catenin antibody showed that more β-Catenin bound to Hes1 promoter in p21G2TKO cells. N = 3, *, p < 0.05; **, p < 0.01.

Article Snippet: After sonication, the supernatant was collected and incubated with ChIP grade protein A magnetic beads and anti-E2F1 antibody (Novus, used 2 μg for 20 μg of chromatin DNA), or anti-β-Catenin antibody (non-phosphorylated (active), CST, used 2 μg for 20 μg of chromatin DNA) overnight at 4 °C.

Techniques: Activation Assay, Inhibition, Co-Immunoprecipitation Assay, Binding Assay, Activity Assay

Journal: Cell death discovery

Article Title: p21 Regulates Wnt-Notch balance via DREAM/MMB/Rb-E2F1 and maintains intestinal stem cell homeostasis.

doi: 10.1038/s41420-024-02192-z

Figure Lengend Snippet: Fig. 5 Overexpression of p21 rescued the imbalance of Wnt-Notch, and shifted the DREAM/MMB/Rb-E2F1 complex towards its inhibitory function. A Overexpression of p21 in p21G2TKO cells restored the expression of Wnt3, β-Catenin proteins, and suppressed the expression of Nocth1, Hes1, and Rbpj. B Overexpression of p21 suppressed the expression of MMB/E2F1 proteins and restored the inhibition DREAM/Rb proteins. C The MMB down-stream proteins and cell cycle related proteins were also suppressed by p21 overexpression. D Immunofluorescence staining indicated the up-regulation of Wnt3 and β-Catenin, and suppression of Nocth1, Hes1 protein level after overexpression of p21.

Article Snippet: After sonication, the supernatant was collected and incubated with ChIP grade protein A magnetic beads and anti-E2F1 antibody (Novus, used 2 μg for 20 μg of chromatin DNA), or anti-β-Catenin antibody (non-phosphorylated (active), CST, used 2 μg for 20 μg of chromatin DNA) overnight at 4 °C.

Techniques: Over Expression, Expressing, Inhibition, Staining

Journal: Molecular Oncology

Article Title: Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

doi: 10.1002/1878-0261.13702

Figure Lengend Snippet: High expression of PRMT5, E2F1 and MYCN correlates with poor prognosis in neuroblastoma and sensitivity to a PRMT5‐specific inhibitor (T1‐44). (A) A representative Immunoblot ( n = 3) displaying protein levels of PRMT5, E2F1 and MYCN in a panel of neuroblastoma cell lines grouped according to their E2F1 and MYCN expression levels. LN‐CAP prostate cancer cells were included as a control cell line without MYCN amplification. IC 50 values representing sensitivity of each cell line to PRMT5 inhibitor T1‐44 are displayed at the bottom of the immunoblot. (B) FACs analysis of three sensitive cell lines and two insensitive cell lines measuring the Sub‐G 1 fraction of cells stained with propidium iodide following treatment with increasing concentrations of T1‐44 (8 n m –1 μ m ) for 144 h. Results are the mean values ± SD; One‐way ANOVA with Tukey's multiple comparison test performed between CHP‐134 and GI‐ME‐N cells at 200 n m concentration: adjusted P < 0.0001; n = 3 independent experiments (each with three technical replicates). (C) Overall (above) and event‐free (below) patient survival probability in stage 1‐stage 4 neuroblastoma patients in the Kocak databases ( n = 634 patients with survival data available out of the cohort of 649) with respect to the PRMT5 , E2F1 and MYCN mRNA expression in these tumours. P values were calculated with a log‐rank test for survival curves. (D) (i) Correlation of PRMT5 ( P < 6.31e‐03 between stage 1 and stage 4 neuroblastoma), E2F1 ( P < 6.81e‐17 between stage 1 and stage 4 neuroblastoma), MYCN ( P < 1.69e‐11 between stage 1 and stage 4), and INSS (International Neuroblastoma Staging System) neuroblastoma stage 1 ( n = 153), stage 2 (113), stage 3 ( n = 93) and stage 4 ( n = 214). Kruskal–Wallis with Dunn's multiple comparisons test was used to determine P values, which were corrected with the Bonferonni correction method. (ii) Box plot correlational analysis of PRMT5 ( P < 3.98e‐26) and E2F1 ( P < 2.07e‐15) mRNA in the MYCN amplified ( n = 550) and non‐ MYCN amplified ( n = 93) neuroblastoma tumours. P values were calculated with a two‐sided Wilcoxon rank sum test for box‐plots. Box‐plot centre represents mean, the box represents SD, and whiskers represent minimum and maximum.

Article Snippet: Stable GFP‐E2F1 expressing CHP‐134 cell lines were generated by transfecting a pMaX‐GFP vector containing a full‐length E2F1 insert (plasmid #16007, Addgene, Watertown, MA, USA) into CHP‐134 E2F1 Cr (CRISPR) cells and selecting with G418.

Techniques: Expressing, Western Blot, Control, Amplification, Staining, Comparison, Concentration Assay

Journal: Molecular Oncology

Article Title: Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

doi: 10.1002/1878-0261.13702

Figure Lengend Snippet: PRMT5 inhibition by T1‐44 deregulates alternative splicing events in sensitive cell lines. (A) (i) A heat map displaying significant differences in the differentially expressed genes (DEGs) between T1‐44 treated and DMSO control treated CHP‐134 cells. Normalised variance stabilising transformation (vst)‐transformed gene expression values corresponding to significantly differentially expressed genes (FDR < 0.01, log2(FC) > 1) were mean‐centered by rows. Each row of the heatmap represents transformed expression values of one differentially expressed gene (DEG) across all samples (blue: low expression; red: high expression). These data were generated from three independent biological samples ( n = 3). (ii) Functional characterisation of CHP‐134 differentially expressed genes using pathfindR. KEGG terms related to cell cycle, ribosome and spliceosome are highlighted in red ( https://www.genome.jp/kegg‐bin/show_pathway?ko03040 ). (B) Distinct differential gene expression changes between T1‐44 treated and DMSO control treated CHP‐134 (E2F1 and MYCN high) and GI‐ME‐N (E2F1 and MYCN low) cell lines are represented in graphical form as a count for upregulated and downregulated genes ( P adj < 0.01, log2FC > 1) ( n = 3). (C) A heatmap displaying the normalised variance stabilising transformation (vst)‐transformed expression values from each biological replicate of T1‐44 or DMSO‐treated CHP‐134 and GI‐ME‐N cells used in the RNA‐seq analysis for 16 E2F1 target genes of interest (selected from RNA splicing factors and classical E2F1 target genes involved in cell cycle progression) ( n = 3). (D) (i) Differential changes in splicing between T1‐44 treated and DMSO control treated CHP‐134 and GI‐ME‐N cell lines are displayed as a heatmap of PSI values (Ψ, per cent spliced in) for all significant differential splicing events (FDR < 0.01). These data were generated from three independent biological samples. (ii) The bar chart displays the statistically significant differential splicing events in each cell line after treatment with T1‐44. The total number of these splicing changes corresponding to different types of splicing event is displayed in different colours. A3SS, alternative 3′ splice; A5SS, alternative 5′ splice site; MXE, mutually exclusive exons; RI, retained intron; SE, skipped/cassette exon. (iii) Venn diagrams showing the overlap between differential splicing events or differentially spliced genes identified in each of the cell lines after T1‐44 treatment.

Article Snippet: Stable GFP‐E2F1 expressing CHP‐134 cell lines were generated by transfecting a pMaX‐GFP vector containing a full‐length E2F1 insert (plasmid #16007, Addgene, Watertown, MA, USA) into CHP‐134 E2F1 Cr (CRISPR) cells and selecting with G418.

Techniques: Inhibition, Alternative Splicing, Control, Transformation Assay, Gene Expression, Expressing, Generated, Functional Assay, RNA Sequencing

Journal: Molecular Oncology

Article Title: Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

doi: 10.1002/1878-0261.13702

Figure Lengend Snippet: Splicing factors are transcriptional targets for E2F1 and MYCN in neuroblastoma cell lines. (A) The mRNA expression level of splicing factors CPSF3 , SRSF1 and HNRNPA3 in CHP‐134 and GI‐ME‐N cell lines. Significance was calculated with an unpaired t ‐test. Results are the mean values ± SD; n = 3 independent experiments (each with three technical replicates). Immunoblot showing the protein expression levels of MYCN and E2F1 in the CHP‐134 and GI‐ME‐N cell lines. GAPDH served as a loading control for this experiment. (B, C) ChIP assays denoting the binding affinity of MYCN and the E2F1 transcription factors on the promoter region of the splicing factor genes CPSF3 , HNRNPA3 and SRSF1 in both CHP‐134 and GI‐ME‐N cell lines. Results represent mean percentage enrichment values ± SD; Significance was calculated with one‐way ANOVA with Tukey's multiple comparison test; ( n = 3 independent experiments, each with three technical replicates). PARP2 and CDC6 served as a positive control for the binding of MYCN and E2F1 respectively. (D) The mRNA expression levels of splicing factors CPSF3 , HNRNPA3 and SRSF3 in a MYCN SHEP‐21N Tet‐off inducible cell line treated with (+DOX) or without (−DOX) doxycycline; Significance was calculated with an unpaired t ‐test. Results represent mean expression values ± SD; n = 3 independent experiments (each with three technical replicates). A Representative immunoblot ( n = 3 independent experiments) showing the protein expression levels of MYCN and E2F1 in SHEP‐21N (−DOX) MYCN overexpressing cells and the SHEP‐21N (+DOX) non‐MYCN‐expressing cells following treatment with doxycycline for 72 h. GAPDH served as a loading control for this experiment. (E) Chromatin immunoprecipitation (ChIP) assays denoting the binding affinity of the MYCN transcription factor on the promoter region of the splicing factor genes CPSF3 , HNRNPA3 and SRSF3 in both SHEP‐21N cells overexpressing MYCN (−DOX) and in cells with decreased MYCN expression (+DOX for 72 h). Results represent mean percentage enrichment values ± SD; Significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). PARP2 served as a positive control for the binding of MYCN. (F) Chromatin immunoprecipitation (ChIP) assays denoting the binding affinity of the E2F1 transcription factor on the promoter region of the splicing factor genes CPSF3 , HNRNPA3 and SRSF3 in both SHEP‐21N cells overexpressing MYCN (−DOX) and in cells with decreased MYCN expression (+DOX for 72 h). Results represent mean percentage enrichment values ± SD; Significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). CDC6 served as a positive control for the binding of E2F1.

Article Snippet: Stable GFP‐E2F1 expressing CHP‐134 cell lines were generated by transfecting a pMaX‐GFP vector containing a full‐length E2F1 insert (plasmid #16007, Addgene, Watertown, MA, USA) into CHP‐134 E2F1 Cr (CRISPR) cells and selecting with G418.

Techniques: Expressing, Western Blot, Control, Binding Assay, Comparison, Positive Control, Chromatin Immunoprecipitation

Journal: Molecular Oncology

Article Title: Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

doi: 10.1002/1878-0261.13702

Figure Lengend Snippet: Changes in the differential splicing events of the apoptotic genes, DIABLO , BCL2L11 , ACIN1 , and CFLAR upon treatment with T1‐44 in CHP‐134. (A) mRNA expression levels of DIABLO , BCL2L11 , ACIN1 and CFLAR in CHP‐134 and GI‐M‐EN cells treated with T1‐44 (200 n m ) or DMSO for 72 h. Results represent the mean expression values ± SD; significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). (B) A representative Immunoblot ( n = 3 independent experiments) displaying symmetric dimethylation (SDMe) protein levels in CHP‐134 and GI‐ME‐N cells treated with and without T1‐44 (200 n m ) for 72 h. GAPDH served as a loading control for these experiments. (C) Changes in the differential splicing events of the apoptotic genes DIABLO , BCL2L11 , ACIN1 , and CFLAR upon treatment of CHP‐134 and GI‐ME‐N cell lines with T1‐44 for 72 h. Results represent mean inclusion/exclusion ratios of the skipped exons ± SD; significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). Above each graph is a schematic representation of the differential splice events in each gene. Exon structure for the skipped exon and flanking exons is displayed, with untranslated regions (UTR) and the open reading frame (ORF) marked in grey and blue respectively. The predicted impact of each splicing event on the ORF and derived protein sequence (displayed in red) are also included. If the skipped exon is known to encode for an amino acid sequence contributing to an annotated protein domain, this is also indicated. DED1, Death effector domain 1; MTS, mitochondrial targeting signal. (D) A representative Immunoblot ( n = 3 independent experiments) displaying the protein levels of E2F1, PRMT5, MYCN and symmetric dimethylation (SDMe) in wild‐type E2F1 (WT E2F1) and CRISPR E2F1 knockout (E2F1 Cr) CHP‐134 cell lines treated for 144 h with four increasing T1‐44 concentrations (8 n m , 40 n m , 200 n m and 1 μ m ). ß‐actin served as a loading control and SDMe served as a control for PRMT5 activity. (E) Changes in the differential splicing of the apoptotic genes DIABLO , BCL2L11 , ACIN1 and CFLAR upon treatment of wild‐type (WT) E2F1 and E2F1 CRISPR (Cr) CHP‐134 cell lines with T1‐44 for 72 h. Results represent the mean inclusion/exclusion ratios of the skipped exons ± SD; significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). (F) mRNA expression levels of DIABLO , BCL2L11 , ACIN1 and CFLAR in wild‐type (WT) E2F1 and E2F1 CRISPR (Cr) CHP‐134 cells treated with T1‐44 or DMSO for 72 h. Results represent the mean expression values ± SD; significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates).

Article Snippet: Stable GFP‐E2F1 expressing CHP‐134 cell lines were generated by transfecting a pMaX‐GFP vector containing a full‐length E2F1 insert (plasmid #16007, Addgene, Watertown, MA, USA) into CHP‐134 E2F1 Cr (CRISPR) cells and selecting with G418.

Techniques: Expressing, Comparison, Western Blot, Control, Derivative Assay, Sequencing, CRISPR, Knock-Out, Activity Assay

Journal: Molecular Oncology

Article Title: Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

doi: 10.1002/1878-0261.13702

Figure Lengend Snippet: The regulation of alternative splicing events following PRMT5 inhibition by T1‐44 is E2F1 dependent. (A) (i) A heat map displaying significant differentially expressed genes (DEGs) between wild‐type (WT) E2F1 and E2F1 CRISPR (Cr) CHP‐134 cells treated with T1‐44 or control DMSO. Normalised variance stabilising transformation (vst)‐transformed gene expression values corresponding to significantly expressed genes (FDR < 0.01 and log 2 (FC) > 1.25) were mean‐centered by rows. Each row of the heatmap represents transformed expression values of one DEG across all samples (blue: low expression; red: high expression). These data were generated from three independent biological samples ( n = 3). (ii) Immunoblot to display protein levels of E2F1 in the WT E2F1 and E2F1 Cr CHP‐134 cell lines. β‐Actin serves as a loading control for this experiment. (B) A heat map displaying values of PSI (Ψ; per cent spliced in) in wild‐type (WT) E2F1 and E2F1 CRISPR (Cr) CHP‐134 cells treated with T1‐44, corresponding to statistically significant differential splicing event changes (FDR < 0.01) with respect to the WT E2F1 CHP‐134 cell line treated with DMSO. Yellow colour represents low PSI values, and blue colour represents the high PSI values. These data were generated from three independent biological samples ( n = 3). (C) The bar chart displays the statistically significant differential splicing events for each treatment, as compared to wild‐type (WT) E2F1 CHP‐134 cells treated with DMSO. The total number of these splicing changes corresponding to different types of splicing events is displayed in different colours. A3SS, alternative 3′ splice; A5SS, alternative 5′ splice site; MXE, mutually exclusive exons; RI, retained intron; SE, skipped/cassette exon. These data were generated from three independent biological samples ( n = 3).

Article Snippet: Stable GFP‐E2F1 expressing CHP‐134 cell lines were generated by transfecting a pMaX‐GFP vector containing a full‐length E2F1 insert (plasmid #16007, Addgene, Watertown, MA, USA) into CHP‐134 E2F1 Cr (CRISPR) cells and selecting with G418.

Techniques: Alternative Splicing, Inhibition, CRISPR, Control, Transformation Assay, Gene Expression, Expressing, Generated, Western Blot

Journal: Molecular Oncology

Article Title: Sustained cancer‐relevant alternative RNA splicing events driven by PRMT5 in high‐risk neuroblastoma

doi: 10.1002/1878-0261.13702

Figure Lengend Snippet: Sensitivity to PRMT5 inhibition by T1‐44 is E2F1 dependent. (A) CHP‐134 cells were treated with 200 n m T1‐44 for 48 h prior to immunoprecipitation of cell extracts with E2F1 antibodies, or non‐specific IgG. The resulting immuno‐precipitates were analysed by immunoblot using anti‐E2F1 antibodies. The blot was also probed with anti‐symmetric dimethylation (SDMe) antibodies (E2F1Me), looking specifically for a band that migrated at the same molecular weight as E2F1 ( n = 3). (B) IC 50 curves for wild‐type (WT) E2F1 and E2F1 CRISPR (Cr) CHP‐134 cell lines treated for 144 h with increasing T1‐44 concentrations (10 −5 –10 5 n m ), with DMSO serving as the untreated control. IC 50 curves were determined by nonlinear regression (curve fit) using log 10 (inhibitor) concentration versus response (three parameters) model in graphpad prism . Results represent mean percentage survival values ± SD per compound concentration; n = 3 independent experiments (each with three technical replicates). (C) Representative Immunoblot ( n = 3 independent experiments) showing E2F1 expression in wild‐type (WT) E2F1 and E2F1 CRISPR (Cr) CHP‐134 cells, and in E2F1 Cr cells stably expressing ectopic wild‐type GFP‐tagged E2F1 (E2F1 Cr + GFP‐E2F1). ß‐actin served as a loading control for this experiment. (D) IC 50 curves for the wild‐type (WT) E2F1, E2F1 CRISPR (Cr) and the E2F1 Cr with stably expressed full‐length GFP‐E2F1 cell lines treated for 144 h with increasing T1‐44 concentrations (10 −5 –10 5 n m ). DMSO treatment served as the untreated control. IC 50 curves were determined by nonlinear regression (curve fit) using log 10 (inhibitor) concentration versus response (three parameters) model in graphpad prism Results represent mean percentage survival values ± SD per compound concentration; n = 3 independent experiments (each with three technical replicates). (E) A representative immunoblot of n = 3 independent experiments displaying symmetric dimethylation (SDMe) protein levels in wild‐type (WT) E2F1, E2F1 CRISPR (Cr) and the E2F1 Cr cells expressing full‐length GFP‐E2F1, treated with T1‐44 (200 n m ) or DMSO for 72 h. ß‐Actin served as a loading control for these experiments. (F) Changes in the differential splicing events of the apoptotic genes DIABLO , BCL2L11 , ACIN1 , and CFLAR upon the treatment of wild‐type (WT) E2F1, E2F1 CRISPR (Cr) and the E2F1 Cr cells stably expressing full‐length GFP‐E2F1 treated with T1‐44 or DMSO for 72 h. Results represent the mean inclusion/exclusion ratios for the skipped exons ± SD; significance was calculated with a one‐way ANOVA with Tukey's multiple comparison test; n = 3 independent experiments (each with three technical replicates). (G) A representative immunoblot of n = 3 independent experiments displaying the over‐expression of FLAG‐tagged DIABLO plasmid constructs lacking (EXON2 − ) or containing exon2 (EXON2 + ) sequence in their open reading frames (ORFs), in wild‐type CHP‐134 cells. The cells are compared against parental CHP‐134 cells that express only endogenous DIABLO. ß‐Actin served as a loading control for this experiment. (H) IC 50 curves of ectopically expressing DIABLO (EXON 2 + and EXON 2 − ) CHP‐134 cell lines treated for 144 h with increasing T1‐44 concentrations (10 −5 –10 5 n m ). DMSO treatment served as the untreated controls. Curves were determined by nonlinear regression (curve fit) using log 10 (inhibitor) concentration versus response (three parameters) model in graphpad prism Results represent mean percentage survival values ± SD per compound concentration; n = 3 independent experiments (each with three technical replicates). (I) Model describing the interplay between MYCN, E2F1 and PRMT5 activity, which regulate the expression of downstream target genes including those encoding for splicing factors and RNA‐binding proteins such as members of the SRSF, HNRNP, and CPSF families. These splicing factors then subsequently regulate the alternative splicing of mRNAs, including those regulated by PRMT5 activity and derived from MYCN and E2F1 target genes, for example apoptotic regulators such as DIABLO, to produce protein isoforms that are defective at driving apoptosis. In cell lines expressing low levels of MYCN, E2F1 and PRMT5, the splicing programme is altered to give rise to protein isoforms that are more efficient at driving functional apoptosis. PRMT5 inhibition also causes a consequent shift in spliced isoforms expressed from the apoptotic genes towards variants that preferentially drive apoptosis. This in part might explain the increased sensitivity to T1‐44 observed in neuroblastoma cell lines expressing high levels of MYCN, PRMT5 and E2F1.

Article Snippet: Stable GFP‐E2F1 expressing CHP‐134 cell lines were generated by transfecting a pMaX‐GFP vector containing a full‐length E2F1 insert (plasmid #16007, Addgene, Watertown, MA, USA) into CHP‐134 E2F1 Cr (CRISPR) cells and selecting with G418.

Techniques: Inhibition, Immunoprecipitation, Western Blot, Molecular Weight, CRISPR, Control, Concentration Assay, Expressing, Stable Transfection, Comparison, Over Expression, Plasmid Preparation, Construct, Sequencing, Activity Assay, RNA Binding Assay, Alternative Splicing, Derivative Assay, Functional Assay

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: miR-9-3p down-regulates E2F1 by directly targeting its 3′ UTR. (A) WT 3′ UTR of E2F1, E2F3, MDM2, or PDK1 or mutant 3′ UTR of E2F1 mRNA was cloned into pMIR-REPORT downstream of Firefly luciferase, and the resulting plasmid was designated pMIR-3′-UTR-E2F1, pMIR-3′-UTR-E2F3, pMIR-3′-UTR-MDM2, pMIR-3′-UTR-PDK1, or pMIR-3′-UTR-E2F1 mutant, respectively (the underlines represent the mutated seed regions). (B) Nontargeted or miR-9-3p mimic was transfected into MCF-7 cells together with pMIR-3′-UTR-E2F1, pMIR-3′-UTR-E2F3, pMIR-3′-UTR-MDM2, pMIR-3′-UTR-PDK1, or pMIR-3′-UTR-E2F1 mutant and a control Renilla luciferase expression vector. 60 h after transfection, cells were harvested and assayed for relative luciferase units. (C) MCF-7 cells were transfected with nontargeted miRNA, miR-9-3p mimic, or miR-9-3p antagomir. 60 h after transfection, cells were harvested, and E2F1 expression was analyzed using RT-qPCR at the mRNA level (left) or Western blotting at the protein level (right). (D, top) MCF-7 cells were treated with PP242 or rapamycin as indicated and analyzed for E2F1 expression as described in C. (Bottom) 36 h after transfection with a miR-9-3p antagomir gradient, MCF-7 cells were treated with 200-nM PP242 for an additional 24 h, and E2F1 expression was analyzed via Western blotting. Error bars represent mean values ± SEM. ns, not significant.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Mutagenesis, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: miR-9-3p acts downstream of mTORC2 and triggers apoptosis by targeting E2F1. (A) MCF-7 cells were transfected with a pool of two siRNAs (1:1) targeting different regions of E2F1 mRNA. After 36 h, cells were serum starved or treated with 400-µM 5-FU for an additional 24 h, harvested, and labeled with Annexin V–FITC and propidium iodide for analysis of apoptosis. The data shown are from a single representative experiment out of three repeats. (B) MCF-7 cells were transfected separately with the two siRNAs and treated as in A, followed by trypan blue staining (top) or Western blotting (bottom). (C) MCF-7 cells were transfected with vehicle or WT E2F1 in the absence or presence of PP242, as indicated. 12 h after transfection, cells were serum starved for an additional 24 h and harvested for either trypan blue staining (top) or Western blotting (bottom). (D) MCF-7 cells were sequentially transfected with Rictor siRNA and WT E2F1. After 24 h of serum starvation, the effects of ectopic E2F1 expression on Rictor knockdown were monitored via trypan blue staining (top) and Western blotting (bottom). (E) MCF-7 cells were sequentially transfected with miR-9-3p and E2F1, followed by assay as described in C. Error bars represent mean values ± SEM. C, control; NC, negative control.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Transfection, Labeling, Staining, Western Blot, Expressing, Knockdown, Control, Negative Control

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: mTORC2 mediates pri-miR-9-2 / miR-9-3p /E2F1 signaling and apoptosis via c-Myc. (A) The effect of silencing c-Myc on the pri-miR-9-2 / miR-9-3p level was assayed using RT-qPCR (left and middle). Protein expression of c-Myc was analyzed via Western blotting (right). (B) MCF-7 cells were treated with PP242 or rapamycin (left) or transfected with two different siRNAs for Rictor and Raptor (right) as indicated, and their effects on c-Myc expression were monitored using Western blotting. (C) MCF-7 cells were treated with 200-nM PP242 or 10-nM rapamycin for 24 h and subjected to chromatin extraction. Sheared chromatin was immunoprecipitated with a c-Myc antibody and the two binding promoter regions of hsa-miR-9-2 (one of three miR-9-3p –coding DNAs) augmented using RT-qPCR. (D and E) The effect of c-Myc silencing on the pri-miR-9-2 / miR-9-3p level regulated by PP242 (D) or Rictor knockdown (E) was analyzed using RT-qPCR. (F–I) The effect of c-Myc silencing on apoptosis induced upon Rictor depletion or PP242 treatment was detected with a light microscope (F), trypan blue staining (G), or Western blotting (H and I), as indicated. Bar, 50 µm. (J) In MDA-MB-231 cells, the effect of c-Myc silencing on apoptosis induced upon 200-nM PP242 treatment (left) or Rictor depletion (right) was detected with Western blot analysis of PARP cleavage. C, control; NC, negative control; TSS, transcription start site. Error bars represent mean values ± SEM.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Extraction, Immunoprecipitation, Binding Assay, Knockdown, Light Microscopy, Staining, Control, Negative Control

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: mTORC2 modulates c-Myc/ miR-9-3p /E2F1 to promote survival in tumor xenografts and a mouse genetic model. (A and B) Images (A) and tumor growth curves (B) of MDA-MB-231 xenografts in BALB/c nude mice treated with vehicle, rapamycin, or PP242 daily by gavage. Treatment groups comprised five mice each. Each data point signifies the estimated tumor areas. Bar, 1 in. (C) Tumor weights of MDA-MB-231 xenografts in nude mice treated with vehicle, rapamycin, or PP242. (D) MDA-MB-231 xenografts were subjected to TUNEL labeling, with the percentage of apoptotic cells calculated under a light microscope. ***, P < 0.001 for comparison of PP242 therapy versus control therapy. Bars, 50 µm. (E and F) MDA-MB-231 xenografts were analyzed for expression of the indicated proteins via Western blotting (E) or miR-9-3p via RT-qPCR (F). (G–J) B lymphocytes from mice conditionally deficient in the Rictor gene were harvested and analyzed for cell death rate with Annexin V labeling (G) and trypan blue staining (H), expression of miR-9-3p with RT-qPCR (I), or cleavage of PARP and expression of c-Myc and E2F1 using Western blotting (J). The figures shown are from a single representative experiment out of three repeats. Error bars represent mean values ± SEM.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: TUNEL Assay, Labeling, Light Microscopy, Comparison, Control, Expressing, Western Blot, Quantitative RT-PCR, Staining

Journal: The Journal of Cell Biology

Article Title: mTORC2 promotes cell survival through c-Myc–dependent up-regulation of E2F1

doi: 10.1083/jcb.201411128

Figure Lengend Snippet: Antagomir-9-3p restores AZD8055-suppressed growth and survival of tumor xenografts. (A and B) BALB/c nude mice were treated with captisol or AZD8055 by gavage and intratumorally injected with antagomirs of NC and miR-9-3p . Each treatment group comprised eight mice. (A) Representative images of mice (top) and MDA-MB-231 xenografts (bottom). (B) Tumor growth curves (left) and tumor weights (right) of MDA-MB-231 xenografts. (C and D) MDA-MB-231 xenografts treated as described in A were either analyzed for the indicated proteins by Western blotting (C) or subjected to TUNEL labeling (D). Images of DAB/hematoxylin staining under a light microscope (40×) are shown. Bar, 50 µm. Arrowheads indicate apoptotic cells. (E) A schematic diagram illustrates the currently defined mTORC2–CIP2A–PP2A–c-Myc– miR-9-3p –E2F1 pathway. Error bars represent mean values ± SEM. NC, negative control.

Article Snippet: Plasmid myc-Rictor (plasmid 1860; ) coding for human Rictor fused to Myc in pRK-5 and plasmid pSG5L HA E2F1 (plasmid 10736; ) coding for human E2F1 fused to HA in pSG5L were obtained from Addgene.

Techniques: Injection, Western Blot, TUNEL Assay, Labeling, Staining, Light Microscopy, Negative Control

Journal: The Journal of urology

Article Title: A Murine Model of K-RAS and β-Catenin Induced Renal Tumors Expresses High Levels of E2F1 and Resembles Human Wilms Tumor.

doi: 10.1016/j.juro.2015.04.090

Figure Lengend Snippet: Figure 5. E2F1 is up-regulated in murine KrasG12D/CatnbDex3 kidneys. Mice were bred and sacrificed at ages 15 to 20 weeks. Kidneys were snap frozen, RNA was extracted and RT-PCR was performed for E2F1 (A). Results were compared to kidneys from controls and analyzed across groups using 2eDDC T .16 BCat, b-catenin. Asterisk indicates p <0.05. Whole protein lysates were extracted and tested by immunoblot (B). Ctrl, control. Formalin fixed, paraffin embedded kidneys were tested for E2F1 expression by IHC (C to F ). Reduced from 20 (C to F ).

Article Snippet: The antibodies used for IHC and/or immunoblotting were c-Myc (Epitomics ), Actin (Sigma-Aldrich ), Axin2 (Abcam ), E2F1 (Atlas Antibodies, Stockholm, Sweden), survivin and Cyclin D1 (Cell Signaling Technology ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Expressing

Journal: The Journal of urology

Article Title: A Murine Model of K-RAS and β-Catenin Induced Renal Tumors Expresses High Levels of E2F1 and Resembles Human Wilms Tumor.

doi: 10.1016/j.juro.2015.04.090

Figure Lengend Snippet: Figure 6. E2F1 is expressed at high levels in most human WTs. IHC was performed for E2F1 on TMA of 32 human WTs. Strong nuclear staining was seen in 30 of 32 WTs (94%) and seen in blastemal elements at low power (A) and high power (B), in stromal elements (asterisk, B), and epithelial elements at low power (C ) and high power (D). Reduced from 20 (A and C ) and 40 (B and D).

Article Snippet: The antibodies used for IHC and/or immunoblotting were c-Myc (Epitomics ), Actin (Sigma-Aldrich ), Axin2 (Abcam ), E2F1 (Atlas Antibodies, Stockholm, Sweden), survivin and Cyclin D1 (Cell Signaling Technology ).

Techniques: Staining