Journal: Molecular Oncology

Article Title: The epithelial splicing regulator ESRP2 is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

doi: 10.1002/1878-0261.13101

Figure Lengend Snippet: Identification of ESRP2 as a candidate hypermethylated gene. (A) Venn diagram showing filtering of the 225 methylated genes that were identified by methyl CpG immunoprecipitation (MCIP), firstly by negative selection for genes that are polycomb repressive complex (PRC) marked in embryonic stem cells and secondly by positive selection for genes that are upregulated in the renal vesicle during kidney development. The full list of methylated genes and filtered lists are shown in Table . dev., development. (B) Gene Ontology analysis of the 225 methylated genes. Only categories with a fold enrichment > 3 are shown; Table for full results. (C) Bar chart of CHST2 , KIT , PTTG1IP and ESRP2 DNA methylation. Controls (Blank, Me 0% (unmethylated DNA control), Me 100% (fully methylated DNA control)), Cell lines (Wilms tumour cell lines, n = 2), FK (foetal kidney, n = 4), NK (normal kidney, n = 2) and Wilms tumours ( n = 15). DNA methylation was assayed by pyrosequencing; Table for pyrosequencing primers. (D) Alternative splicing of ENAH exon 11A was analysed by RT‐PCR followed by agarose gel electrophoresis in 2 WT cell lines (Wit49 and 17.94), FK, NK and 4 WTs. Representative of n = 3.

Article Snippet: DNA was purified by phenol/chloroform extraction, bisulphite converted (EZ DNA Methylation Gold kit; Zymo Research), amplified using a PyroMark PCR kit (Qiagen) and pyrosequenced on a PyroMark Q96 instrument (Qiagen), using primers listed in Table .

Techniques: Methylation, Immunoprecipitation, Selection, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Molecular Oncology

Article Title: The epithelial splicing regulator ESRP2 is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

doi: 10.1002/1878-0261.13101

Figure Lengend Snippet: ESRP2 is hypermethylated in Wilms tumours and nephrogenic rests. (A) Dot‐boxplot of ESRP2 DNA methylation in the BCH cohort. NT (normal tissue) n = 8 (4 NK (normal kidney) and 4 FK (foetal kidney), WT (Wilms tumour) n = 65, P value from t ‐test. (B) Dot‐boxplot of ESRP2 and ESRP1 DNA methylation in the RMH cohort. ESRP2 : NT n = 18 (all NK), WT n = 73; ESRP1 : NT n = 15 (all NK), WT n = 69, P values from t ‐test. (C) Dot‐boxplot of ESRP2 and ESRP1 DNA methylation in dataset GSE59157 . NT n = 36 (all NK), WT n = 37, P values from t ‐test. (D) RASSF1A and ESRP2 methylation in nephrogenic rests in the BCH cohort. Two sets of matched NK, NR (nephrogenic rest) and WT are shown. (E) Dot‐boxplot of RASSF1A and ESRP2 methylation in nephrogenic rests in the GSE59157 data set. 17 sets of matched NK, NR and WT are shown for RASSF1A methylation and 13 sets for ESRP2 methylation, from individuals where the WT was hypermethylated compared with the matched NK. P values from Tukey’s pairwise test. DNA methylation was assayed by pyrosequencing in A to D and by Illumina Human Methylation 450 bead arrays in E.

Article Snippet: DNA was purified by phenol/chloroform extraction, bisulphite converted (EZ DNA Methylation Gold kit; Zymo Research), amplified using a PyroMark PCR kit (Qiagen) and pyrosequenced on a PyroMark Q96 instrument (Qiagen), using primers listed in Table .

Techniques: DNA Methylation Assay, Methylation

Journal: Molecular Oncology

Article Title: The epithelial splicing regulator ESRP2 is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

doi: 10.1002/1878-0261.13101

Figure Lengend Snippet: ESRP2 expression is repressed in Wilms tumour and regulated by DNA methylation. (A) Dot‐boxplot of ESRP2 RNA expression relative to FK (foetal kidney) in the BCH cohort. NT (normal tissue) n = 6 (3 NK (normal kidney) and 3 FK), WT (Wilms tumour) n = 32, P value from t ‐test. (B) Dot‐boxplot of ESRP2 expression in hypomethylated versus hypermethylated samples in the BCH. Hypomethylated n = 14 (ESRP2 methylation< 25%, Hypo), hypermethylated n = 24 (ESRP2 methylation > 25%, hyper), P value from t ‐test. (C) Dot‐boxplot of ESRP2 and ESRP1 RNA expression relative to NT in the RMH cohort. ESRP2 : NT n = 12 (all NK), WT n = 51; ESRP1 : NT n = 9 (all NK), WT n = 33, P values from t ‐tests. (D) Dot‐boxplot of ESRP2 and ESRP1 RNA expression in the GSE2712 data set. NT n = 3 (all FK), WT n = 18, P values from t ‐tests. (E) 17.94 and Wit49 WT cell lines were treated with 2 µ m Aza (5‐aza‐2’‐deoxycytidine) for six days. ESRP2 RNA levels expressed relative to untreated cells. Results are mean ± SD of n = 3, P values from paired t ‐tests. RNA expression was measured by real‐time qPCR, normalised to endogenous levels of TBP in A, B, C and E, and by Affymetrix Human Genome U133A arrays in D.

Article Snippet: DNA was purified by phenol/chloroform extraction, bisulphite converted (EZ DNA Methylation Gold kit; Zymo Research), amplified using a PyroMark PCR kit (Qiagen) and pyrosequenced on a PyroMark Q96 instrument (Qiagen), using primers listed in Table .

Techniques: Expressing, DNA Methylation Assay, RNA Expression, Methylation

Journal: Molecular Oncology

Article Title: The epithelial splicing regulator ESRP2 is epigenetically repressed by DNA hypermethylation in Wilms tumour and acts as a tumour suppressor

doi: 10.1002/1878-0261.13101

Figure Lengend Snippet: Inducible expression of ESRP2 in a Wilms tumour cell line. V200 is a control WT cell line (transfected with empty vector), and E200L is a WT cell line expressing doxycycline‐inducible ESRP2 (Materials and Methods). (A) ESRP2 RNA expression assayed by qPCR, normalised to endogenous levels of TBP , in V200 and E200L cells after 72‐h doxycycline (Dox) induction, shown as fold induction relative to uninduced V200 cells. Results are mean ±SD of n = 3, P value from paired t ‐test. (B) ESRP2 protein assayed by western blotting in V200 and E200L cells after 72‐h doxycycline induction. Anti‐ESRP2 detected total ESRP2 protein, anti‐FLAG detected vector‐derived ESRP2 and anti‐ACTIN was used as a loading control. Representative of n = 3. (C) Alternative splicing of ENAH exon 11A was analysed by RT‐PCR followed by agarose gel electrophoresis to detect different sized amplicons, in V200 and E200L cells after 72‐h doxycycline induction. Representative of n = 3. (D) Immunofluorescence of E200L cells, stained for FLAG‐tagged ESRP2 (green) and ACTIN (red) in the left‐hand panels, and for nuclear DNA with DAPI (blue) in the right‐hand panels, after 72‐h doxycycline induction (+Dox), or uninduced (‐Dox). Scale bars = 50 µm. (E) Colony‐forming assay of induced (doxycycline‐treated) and uninduced V200 and E200L cells, shown as fold colony numbers compared to uninduced controls after 14 days. Results are mean ± SD of n = 3, P values from paired t ‐test. (F) Cell confluence assay (by IncuCyte), showing growth of induced (doxycycline‐treated) and uninduced V200 and E200L cells. Results are mean ± SD of n = 6, P value at 162 h from paired t ‐test. Representative of n = 3. (G) H: Cell trace violet (CTV) proliferation assay of induced (doxycycline‐treated) and uninduced V200 and E200L cells. (G) CTV staining of triplicates of induced and uninduced cells showing median fluorescence intensity histograms at 6 days of treatment. Red peaks are controls representing staining of cells at day zero. (H) Dot‐boxplot of quantitation of staining at 6 days ( n = 3). P values from paired t ‐tests of log‐transformed values.

Article Snippet: DNA was purified by phenol/chloroform extraction, bisulphite converted (EZ DNA Methylation Gold kit; Zymo Research), amplified using a PyroMark PCR kit (Qiagen) and pyrosequenced on a PyroMark Q96 instrument (Qiagen), using primers listed in Table .

Techniques: Expressing, Transfection, Plasmid Preparation, RNA Expression, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Immunofluorescence, Staining, Proliferation Assay, Fluorescence, Quantitation Assay, Transformation Assay