Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

Article Title: Sma3s: A Three-Step Modular Annotator for Large Sequence Datasets

doi: 10.1093/dnares/dsu001

Figure Lengend Snippet: Number of annotations assigned by each Sma3s module

Article Snippet: We extracted two independent DNA arrays from the GEO database: Affymetrix Murine 11K SubB Array (AC:GPL76) and the Affymetrix Arabidopsis ATH1 Genome Array (AC:GPL198).

Techniques:

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

Article Title: Sma3s: A Three-Step Modular Annotator for Large Sequence Datasets

doi: 10.1093/dnares/dsu001

Figure Lengend Snippet: Annotation of two DNA arrays using Sma3s, Blast2GO and Top-BLAST. The annotation prediction results of mouse and Arabidopsis sequences are shown from Sma3s with all modules, Sma3s with the M3 module only, Blast2GO and Top-BLAST. The corresponding values appear above the bars. Sn, sensitivity; Sp, specificity; SC, sequence coverage; TC, term coverage. This figure appears in colour in the online version of DNA Research .

Article Snippet: We extracted two independent DNA arrays from the GEO database: Affymetrix Murine 11K SubB Array (AC:GPL76) and the Affymetrix Arabidopsis ATH1 Genome Array (AC:GPL198).

Techniques: Sequencing

Journal: Oncogene

Article Title: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

doi: 10.1038/s41388-018-0334-9

Figure Lengend Snippet: a. Schematic illustration showing IDH1 guide RNA (gRNA) sequence and the targeted strand using the “single base editing” technique with a fusion enzyme of catalytically dead Cas9 (dCas9) and cytidine deaminase. b. Sanger sequencing of the PCR products of genomic DNA from an IDH1 wild type clone (WT) and two heterozygous IDH1 R132H/WT clones (C2 and C6). The C2 and C6 showed a double peak with A/G reads at the edited G site. Same approach was used to sequence the PCR product from cDNA of C6 cells. c. Immunoblotting with a specific antibody against IDH1 R132H (Mut IDH1) and an antibody recognizing both wild type and IDH1 R132H confirmed the expression of IDH1 in all three cell lines, but the IDH1 R132H protein was only detected in C2 and C6 cells. d, e. Mass spectrometry was used to measure IDH1 R132H/WT oncometabolite 2-HG in both cell pellets (d) and conditioned media (e) of IDH1 WT and two IDH1 R132H/WT clones. Endogenous IDH1 R132H/WT significantly increased 2-HG level in both the pellets and conditioned media of C2 and C6 cells (error bars stand for standard deviation). f. SVG IDH1 WT cells and IDH1 R132H/WT cells together with human glioblastoma U373 (with mutated TP53 ) and U87 (with wild type TP53 ) cells were treated with mitomycin C at 2.5 μg/ml for 4 hours. RNA was collected for RT-PCR to detect p53 downstream target p21 expression. Following drug treatment, p21 was significantly elevated in U87 cells only, not in SVG IDH1 WT and two IDH1 R132H/WT clones. ***: P <0.001.

Article Snippet: The antibodies used for this study were listed below; most of them from Cell signaling Inc (Danvers, MA) unless otherwise stated: IDH1 mutant-specific (R132H form): Dianova (Hamburg, Germany, DIA-H09); IDH1 (mAb #8137); tenascin C (Millipore); brevican (Abcam); YAP, cyclin A, E1 and CDKs, ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK, Jagged 1, NICD1 and NOTCH1, β –catenin, AKT and phosphor-AKT, IKK β, STAT3 and β-actin (Sigma).

Techniques: Sequencing, Clone Assay, Western Blot, Expressing, Mass Spectrometry, Standard Deviation, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

doi: 10.1038/s41388-018-0334-9

Figure Lengend Snippet: a. Comparison of methylation profiles showing relative DNA methylation distribution for WT and IDH1 R132H/WT cells. Frequency (y-axis) is plotted against DNA methylation level (β, 0–1). Both IDH1 R132H/WT clones showed increased frequency in high methylation levels (0.75–1). b. About 21,647 CpG sites corresponding to 5370 gene promoter regions showed significant methylation alterations, among them ~3500 being hypermethylated, ~1800 hypomethylated in IDH1 R132H/WT cells when compared with control cells. c. Comparison of the methylation profiles between IDH1 R132H/WT SVG and G-CIMP+ tumors. The average methylation level of G-CIMP+ tumor samples was used. The dash grey line indicates the average methylation level of G-CIMP+ in each bin of IDH1 R132H/WT methylation. Pearson’s correlation coefficient (R) is shown in the figure. d. Gene set enrichment analysis (GSEA) of top hypermethylated probes in IDH1 R132H/WT in G-CIMP tumors. Top 2,000 hypermethylated probes in IDH1 R132H/WT relative to wild type SVG (middle black bars) were used to perform GSEA. The green curve is the enrichment curve by comparison of top 2,000 hypermethylated probes in IDH1 R132H/WT to the ranked probes from G-CIMP microarray data (bottom grey bars). The probes from G-CIMP microarray data were ordered based on relative methylation level of CIMP+ to CIMP-. NES, normalized enrichment score. FDR: false discovery rate. e. Global gene expression profiling in IDH1 R132H/WT cells relative to the control cells. A total of 853 genes showed significant changes by IDH1 R132H/WT , 538 being downregulated, 315 being upregulated. f. GSEA of upregulated genes indicated enrichment of PRC targets and cell migration pathways. g. GSEA of upregulated genes indicated enrichment of cell proliferation, extracellular and cell motility pathways. h. Quantitative real-time PCR (RT-PCR) validation of large scale RNA-sequencing analysis showed downregulation of genes involved in extracellular matrix proteins and cell proliferation pathway. All changes are significant. i. A total of 251 genes were identified by overlapping differentially expressed genes and differentially methylated promoter regions from RNA-seq and methylome datasets, respectively. Based on methylation and gene expression changes, four groups of genes were plotted.

Article Snippet: The antibodies used for this study were listed below; most of them from Cell signaling Inc (Danvers, MA) unless otherwise stated: IDH1 mutant-specific (R132H form): Dianova (Hamburg, Germany, DIA-H09); IDH1 (mAb #8137); tenascin C (Millipore); brevican (Abcam); YAP, cyclin A, E1 and CDKs, ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK, Jagged 1, NICD1 and NOTCH1, β –catenin, AKT and phosphor-AKT, IKK β, STAT3 and β-actin (Sigma).

Techniques: Comparison, Methylation, DNA Methylation Assay, Clone Assay, Control, Microarray, Gene Expression, Migration, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, RNA Sequencing

Journal: Oncogene

Article Title: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

doi: 10.1038/s41388-018-0334-9

Figure Lengend Snippet: a. Microphotograph of migrated cells on transwell membrane 24 h after plating. Bar = 100 μm. b. Cell counting of migrated cells at 4 h and 24 h after plating. c. AGI-5198 had no effect on WT cell migration, but completely blocked the pro-migratory effect of IDH1 R132H/WT in C2 cells. d. Cell invasion assays on matrigel-coated transwells also showed that IDH1 R132H/WT dramatically increased cell invasion by more than 7 fold, which was completely reversed by AGI-5198. e. Western blot analysis revealed decrease in extracellular matrix proteins and increase in MMP1 in IDH1 R132H/WT cells. f. Transwell assays of SVG IDH1 WT and IDH1 R132H/WT cells plated on non-coated (non), TNC or collagen-coated surfaces. g. Western blotting analysis showed 2.5–3 fold increase in ITGB4 by IDH1 R132H/WT , which was reversed by AGI-5198 (1.5 μmol/L). h. Compared with control siRNA, ITGB4 specific siRNA downregulated ~50% ITGB protein expression in both SVG IDH1 WT and IDH1 R132H/WT cells. i. ITGB4 knockdown dramatically decreased cell migration induced by IDH1 R132H/WT in transwell assays (***: P <0.001).

Article Snippet: The antibodies used for this study were listed below; most of them from Cell signaling Inc (Danvers, MA) unless otherwise stated: IDH1 mutant-specific (R132H form): Dianova (Hamburg, Germany, DIA-H09); IDH1 (mAb #8137); tenascin C (Millipore); brevican (Abcam); YAP, cyclin A, E1 and CDKs, ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK, Jagged 1, NICD1 and NOTCH1, β –catenin, AKT and phosphor-AKT, IKK β, STAT3 and β-actin (Sigma).

Techniques: Membrane, Cell Counting, Migration, Western Blot, Control, Expressing, Knockdown

Journal: Oncogene

Article Title: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

doi: 10.1038/s41388-018-0334-9

Figure Lengend Snippet: a. Cell growth curves of control, C2 and C6 cells. Cells were plated into 6 well plates at 5×10 4 and counted every two days. At day 2, there was no significant difference in cell growth. At day 4 and day 6, there were ~55–65% less cells in C2 and C6 cultures compared with control. b. Both WT and C6 Cells were incubated with the mutant IDH1 inhibitor AGI-5198 or vehicle DMSO for 2 weeks prior to the cell growth analysis. AGI-5198 had no effect on WT cell growth on day 4, but completely rescued cell number loss in C6 cells. c, d. Cell cycle analysis. Cells were plated at 2×10 5 in 6 cm dishes and grew in 10% serum containing medium for 24 h followed by incubation in 0.1% FCS medium for 36 h. Cells were then replenished with 10% serum and collected at indicated time point after serum addition for cell cycle analysis by flow cytometry/PI staining. Percentage of cells at different cell cycle phase before (0 h, c) and 8 h (d) after serum replenishing were plotted. e. Cells were treated with AGI-5198 and analyzed 8 h after serum repletion followed by cell cycle analysis. f-h. Western blot analysis of cell cycle regulators indicated decrease in CDK6 by IDH1 R132H/WT , which was rescued by AGI-5198 treatment (*: P <0.05, ***: P <0.001).

Article Snippet: The antibodies used for this study were listed below; most of them from Cell signaling Inc (Danvers, MA) unless otherwise stated: IDH1 mutant-specific (R132H form): Dianova (Hamburg, Germany, DIA-H09); IDH1 (mAb #8137); tenascin C (Millipore); brevican (Abcam); YAP, cyclin A, E1 and CDKs, ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK, Jagged 1, NICD1 and NOTCH1, β –catenin, AKT and phosphor-AKT, IKK β, STAT3 and β-actin (Sigma).

Techniques: Control, Incubation, Mutagenesis, Cell Cycle Assay, Flow Cytometry, Staining, Western Blot

Journal: Oncogene

Article Title: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

doi: 10.1038/s41388-018-0334-9

Figure Lengend Snippet: a. Phosphorylation of three MAPK members, namely ERK1/2, JNK and p38, were downregulated by IDH1 R132H/WT . b. Immunoblotting analysis of control, C2 and C6 cells revealed that Jagged 1 and NICD1 was downregulated by IDH1 R132H/WT . The NOTCH1 receptor was not affected by IDH1 R132H/WT . c. The effector of Wnt pathway β-catenin was downregulated in C2 and C6 cells; IDH1 R132H/WT did not alter the AKT pathway. d. YAP protein was highly expressed in WT cells and was downregulated by ~50% in C2 and C6 IDH1 R132H/WT cells. The expression level of TAZ was not decreased by IDH1 R132H/WT . e. AGI-5198 completely rescued IDH1 R132H/WT -mediated decrease in YAP, NICD1 and p-ERK1/2 pathways. f. Cells were treated with PDGF-BB (30 ng/ml) in normal culture medium for 4 days. PDGF-BB significantly increased cell proliferation in C2 and C6 SVG IDH1 R132H/WT cells but not in SVG IDH1 WT cells (*: P <0.05, **: P <0.01).

Article Snippet: The antibodies used for this study were listed below; most of them from Cell signaling Inc (Danvers, MA) unless otherwise stated: IDH1 mutant-specific (R132H form): Dianova (Hamburg, Germany, DIA-H09); IDH1 (mAb #8137); tenascin C (Millipore); brevican (Abcam); YAP, cyclin A, E1 and CDKs, ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK, Jagged 1, NICD1 and NOTCH1, β –catenin, AKT and phosphor-AKT, IKK β, STAT3 and β-actin (Sigma).

Techniques: Phospho-proteomics, Western Blot, Control, Expressing

Journal: Oncogene

Article Title: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

doi: 10.1038/s41388-018-0334-9

Figure Lengend Snippet: a. RT-PCR indicated that the mRNA level of YAP was decreased by more than 50% in C2 and C6 cells when compared with WT cells. b. Immunocytostaining of YAP protein in WT and C2 cells. YAP was expressed in both cytosol and nucleus. IDH1 R132H/WT decreased overall YAP expression in both cytosol and nucleus. Bar = 20 μm. c. YAP mediated the cell growth inhibition effect of IDH1 R132H/WT . WT and C2 cells were transiently transfected with a YAP expression plasmid and a control vector pLex. Cells were counted at 4 days after transfection. d. Identification of downstream targets of YAP in IDH1 R132H/WT cells. C2 cells were transiently transfected with the YAP expressing plasmid and the control vector. After 48 h, proteins were collected for immunoblotting. Elevated YAP increased Jagged 1, but not p-ERK or β-catenin, indicating that the Notch pathway was downstream of YAP. e. The effectors of Notch pathway HES1 and HEY1 were significantly decreased in C2 and C6 clones. YAP downstream target MCL-1 was also downregulated in C2 and C6 cells. f. Cells were treated with the Notch pathway inhibitor DAPT (2 μmol/L) for 30 min prior to YAP or empty vector transfection. Cell number was counted 3 days after transfection. g. Analysis of TCGA data indicated that compared with IDH1 WT tumors, YAP expression level was significantly lower ( P <0.05) in IDH1-mutant glioblastomas (left) and low grade gliomas (LGG, right). h. A survey of 12 primary glioma neurosphere cultures derived from patient samples by immunoblotting with anti-YAP and anti-mutant IDH1 R132H antibodies indicated that IDH1 R132H status was moderately correlated with low YAP protein expression (R 2 =0.38). (*: P <0.05, **: P <0.01, ***: P <0.001).

Article Snippet: The antibodies used for this study were listed below; most of them from Cell signaling Inc (Danvers, MA) unless otherwise stated: IDH1 mutant-specific (R132H form): Dianova (Hamburg, Germany, DIA-H09); IDH1 (mAb #8137); tenascin C (Millipore); brevican (Abcam); YAP, cyclin A, E1 and CDKs, ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK, Jagged 1, NICD1 and NOTCH1, β –catenin, AKT and phosphor-AKT, IKK β, STAT3 and β-actin (Sigma).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Inhibition, Transfection, Plasmid Preparation, Control, Western Blot, Clone Assay, Mutagenesis, Derivative Assay

Journal: Oncogene

Article Title: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

doi: 10.1038/s41388-018-0334-9

Figure Lengend Snippet: Schematic illustration of molecular and cellular events induced by endogenous IDH1 R132H/WT in human astroglial cells with p53 inactivation.

Article Snippet: The antibodies used for this study were listed below; most of them from Cell signaling Inc (Danvers, MA) unless otherwise stated: IDH1 mutant-specific (R132H form): Dianova (Hamburg, Germany, DIA-H09); IDH1 (mAb #8137); tenascin C (Millipore); brevican (Abcam); YAP, cyclin A, E1 and CDKs, ERK and phospho-ERK, p38 and phospho-p38, JNK and phospho-JNK, Jagged 1, NICD1 and NOTCH1, β –catenin, AKT and phosphor-AKT, IKK β, STAT3 and β-actin (Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

doi: 10.1371/journal.pone.0057709

Figure Lengend Snippet: (A) To validate the changes in DNA methylation, the samples were re-analyzed with an Infinium microarray using independently bisulfite modified genomic DNA. The figure represents the number of CpG loci that showed CpG methylation changes with a Δβ-value of ≥0.1 in both experiments. 5-aza-CdR and TSA treated cells were used as positive and negative controls of validation. (B) Quantitative MSP (qMSP) was performed to validate the methylation changes in HCT116. Relative methylation was calculated by normalization of the methylation status of curcumin and 5-aza-CdR treated cells to controls. (C) The Venn diagram shows that CpG methylation changes overlap between HCT116, RKO and HT29 after the treatment with curcumin.

Article Snippet: First, we performed an independent genome-wide methylation microarray analysis (Infinium®) using matched independent bisulfite modified DNA samples from curcumin and control cell lines.

Techniques: DNA Methylation Assay, Microarray, Modification, CpG Methylation Assay, Biomarker Discovery, Methylation