Journal: Epigenetics

Article Title: SFRP1 CpG island methylation locus is associated with renal cell cancer susceptibility and disease recurrence.

doi: 10.4161/epi.19614

Figure Lengend Snippet: Figure 1. Quantitative SFRP1 CGI methylation and gene expression analysis in renal cell lines and normal primary cells. (A) Illustration of SFRP1 CGI structure and relative positions of methylation analysis. (B) Example for quantitative methylation analysis of 11 CpG sites (gray bars) and one control site (narrow gray bar) in the CGI region of the SFRP1 gene using pyrosequencing of bisulfite treated DNA. Here, an average methylation of 35% was determined while a nearly complete bisulfite conversion of 99.6% has been achieved. (C) Quantitative SFRP1 CGI methylation analysis and relative quantitation of SFRP1 mRNA expression in renal cell cancer cell lines and primary cells from normal kidney (RPTEC). (D) Duplicate measurements and Lin’s concordance correlation analysis. The solid line represents the regression line of measurements while the dashed line indicates the line of perfect concordance. Only minute deviations from ideal concordance were observed.

Article Snippet: Colella S, Shen L, Baggerly KA, Issa JP, Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites.

Techniques: Methylation, Gene Expression, Control, Quantitation Assay, Expressing

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: Genome-wide allelic expression imbalance analysis in osteosarcoma. ( A ) A Manhattan plot showing the result of the genome-wide allelic imbalance analysis. The most significant SNPs were found on pig chromosomes 6, 9, 14 and 16. ( B ) Schematic genomic structure of the YAP1-BIRC3 locus on chromosome 9 in pigs. The blue arrow indicates the position of the 9:33044172 A/G SNP in the 3′UTR of BIRC3 . ( C ) cDNA pyrosequencing result for the SNP 9:33044172 A/G in osteosarcoma (os, n = 48) and matched healthy bone (b) samples collected from flTP53 R167H pigs. To test analysis the validity of the pyrosequencing assay, we used DNA samples ( n = 5) extracted from wild-type pigs. *** p < 0.001.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Genome Wide, Expressing, Pyrosequencing Assay

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: YAP1 amplification in p53 deficient osteosarcoma. ( A ) Point plot showing the correlation between 9:33044172 A allele expression and YAP1 copy number. Gray and red points show expression of A allele in bone and OS samples, respectively. Blue points show the measurements in wild-type samples. ( B ) Point plot showing the correlation between 9:33044172 A allele expression and OS ( n = 48) size. ( C ) Point plot showing the correlation between YAP1 copy number and OS ( n = 48) size. ( D ) Quantitative PCR of YAP1 mRNA expression in wild type (wt, n = 5) bones, as well as OS ( n = 48) and matched healthy bone samples from flTP53 R167H pigs. ( E ) Representative Western blot showing YAP1 expression in wild type bone, OS and healthy bone samples from flTP53 R167H pigs. The uncropped Western blots have been shown in . ( F ) Immunohistochemistry staining showing the nuclear location of YAP1 in sections of osteosarcoma from flTP53 R167H pigs. Control samples show staining without the first antibody. Scale bars- 100 μm. (** p < 0.01)

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Amplification, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Control

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: In vitro functional analysis of YAP1 deficiency in p53 deficient primary osteosarcoma cells. ( A ) Sequence analysis showing the result of CRISPR/Cas9 editing of YAP1 in pig OS cells. ( B ) Western blot showing the lack of YAP1 protein in the edited flTP53 R167H OS cells. ( C ) Representative microscopic view showing the morphology of YAP1 −/− /flTP53 R167H OS cells. As a control, flTP53 R167H OS cells were transfected with the GFP control vector (left scale bars, 400μm; right scale bars, 200 μm) ( D ) Proliferation result for YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. ( E ) Representative microscopic images showing a difference in migration and invasion between YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells (scale bars, 200 μm). Quantitative measurement of migration ( F ) and invasion ( G ). ( H ) Immunofluorescence staining for Ki67 and DAPI in YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. ( I ) Quantification rates of the Ki67 positive cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: In Vitro, Functional Assay, Sequencing, CRISPR, Western Blot, Control, Transfection, Plasmid Preparation, Migration, Immunofluorescence, Staining

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: Expression of p53 related genes in YAP1 −/− /flTP53 R167H OS cells. ( A ) RT-PCR result for WRAP53, TP53INP1, p14, p16, RB1, TP63, TP73 in YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. Three independent transfections for each expression vector were performed. NC—negative control. ( B ) Quantitative RT-PCR of p16 mRNA expression. GAPDH mRNA expression was used as a reference. ** p < 0.01. ( C ) Western blot showing lack of p63 expression in YAP1 −/− /flTP53 R167H OS cells.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: DNA methylation analysis of the p16 and Rb1 promoter regions in YAP1 −/− /flTP53 R167H OS cells. ( A ) Pyrosequencing result at 8 CpG sites in the p16 promoter region in YAP1 −/− /flTP53 R167H ( n = 3) and flTP53 R167H ( n = 3) OS cells. ( B ) Pyrosequencing result at 9 CpG sites in the Rb1 promoter region in YAP1 −/− /flTP53 R167H ( n = 3) and flTP53 R167H ( n = 3) OS cells. * p < 0.05.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: DNA Methylation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK1 drives SOX9-mediated chemotherapeutic resistance in gastric cancer

doi: 10.1186/s13046-025-03523-3

Figure Lengend Snippet: CDK1 activates SOX9 in gastric cancer. Ectopic overexpression of CDK1 or empty vector (Ctrl) for 72 h in AGS cells had no effect on SOX9 mRNA expression (A) . While remarkably increasing the SOX9 protein level by western blot analysis (left panel) and increase in nuclear SOX9 following overexpression of CDK1 was detected by Immunofluorescence analysis (right panel) (B) . This was associated with a significant increase in SOX9 luciferase reporter activity (C) . Similar findings were observed in MKN28 cells (D-F). The knockdown of CDK1 in MKN45 cells had no effect on SOX9 mRNA (G). Western blot analysis and immunofluorescence analysis show a decrease in nuclear SOX9 following knockdown of CDK1 in MKN45 cells (H) . This was associated with a significant decrease in SOX9 luciferase reporter activity (I) . The firefly luciferase activity was normalized to renilla luciferase activity. scale bar = 20 μm

Article Snippet: IHC staining procedures were done by employing rabbit anti-human CDK1 antibody (ab133327, Abcam, Cambridge, MA) and SOX9 antibody (37447, Cell Signaling, Danvers, MA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, Luciferase, Activity Assay, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK1 drives SOX9-mediated chemotherapeutic resistance in gastric cancer

doi: 10.1186/s13046-025-03523-3

Figure Lengend Snippet: CDK1 regulates SOX9 levels via miR-145. A Venn diagram showing downregulated miRNAs in human (blue) and mouse (orange) local cohorts (A). miR-145 putative binding sites on the 3’UTR of SOX9 are shown (B). miR-145 expression in human normal and GC tissue samples, P < 0.01. A negative correlation between SOX9 and miR-145 expression levels is detected in human gastric samples (C) . MKN45 cells were treated with dinaciclib (20nM) for 16 h or transfected with siCDK1 or siCtrl for 72 h then qRT-PCR conducted to measure the expression levels of miR-145 (D). MKN45 cells were transfected with miR-145 or the empty vector (Ctrl) for 72 h, miR-145 levels were determined by qRT-PCR (E) and SOX9 protein levels were detected by western blot analysis. A representative β-actin is shown as an internal control in WB (F). AGS cells were transfected with the SOX9-3’UTR reporter and Rluc luciferase constructs with an empty vector (Ctrl) or miR-145 (G) , SOX9-3’UTR mu1, or SOX9-3’UTR mu2 (H) . After 48 h of transfection, firefly luciferase activity in the cell lysates was measured, and reporter activity was normalized to renilla luciferase activity as shown (I)

Article Snippet: IHC staining procedures were done by employing rabbit anti-human CDK1 antibody (ab133327, Abcam, Cambridge, MA) and SOX9 antibody (37447, Cell Signaling, Danvers, MA).

Techniques: Binding Assay, Expressing, Transfection, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Control, Luciferase, Construct, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK1 drives SOX9-mediated chemotherapeutic resistance in gastric cancer

doi: 10.1186/s13046-025-03523-3

Figure Lengend Snippet: CDK1 suppresses miR-145 expression via induction of DNMT1. The miR-145 promoter shows a CpG island with several CpG nucleotides (A). Representative dot plots of methylation status of CpG sites in normal (NG = 7) and tumor (TG = 10) gastric human samples detected by pyrosequencing assay (B). Methylation levels of miR-145 in normal and tumor human gastric samples (C). qRT-PCR analysis shows an increase miR-145 expression in AGS and MKN45, following treatment with 5’Aza for 48 h (D). Representative dot plots of methylation status of CpG sites in control and 5’Aza treated GC cells detected by pyrosequencing assay ( E ). Overexpression of CDK1 (0.25 and 0.5 µg), increases total and pDNMT1 (S145) levels, as compared to control, a representative ß-actin used as an internal control is shown (F) . CDK1 knockdown (siCDK1) showed the opposite effects, as compared to control (siCtrl) (H). DNMT1 enzymatic activity increases following CDK1 overexpression ( G ), with a reverse effect following CDK1 knockdown (I) . Knockdown of DNMT1 in AGS and MKN45 cells led to an increase in miR-145 and decrease SOX9 protein expression ( J - M )

Article Snippet: IHC staining procedures were done by employing rabbit anti-human CDK1 antibody (ab133327, Abcam, Cambridge, MA) and SOX9 antibody (37447, Cell Signaling, Danvers, MA).

Techniques: Expressing, Methylation, Pyrosequencing Assay, Quantitative RT-PCR, Control, Over Expression, Knockdown, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK1 drives SOX9-mediated chemotherapeutic resistance in gastric cancer

doi: 10.1186/s13046-025-03523-3

Figure Lengend Snippet: BCL2L1 is a direct transcription target of SOX9. Graph illustrating the expression level of the top 10 SOX9 signature genes in the TCGA STAD dataset (A). A positive correlation was observed between the expression levels of CDK1 and BCL2L1 (BCL-xL), and between SOX9 and BCL2L1 in the STAD dataset (B) . qRT-PCR analysis of BCL2L1 mRNA expression, following SOX9 knockdown (siSOX9) or control (siCtrl) in AGS (C) and MKN45 cells (D) . Subsequent western blot analysis demonstrates a decrease in BCL-xL protein, following SOX9 knockdown (E) . Predicted SOX9 binding sites motif within the BCL2L1 promoter were identified using the JASPAR database (F) . ChIP-qPCR analysis demonstrates enrichment of P1 and P3 binding sites, normalized to the IgG control (G) . AGS cells were transfected with SOX9 plasmid and BCL2L1 full length promoter (FL) reporter, along with luciferase reporter plasmids containing mutated SOX9 binding sites (muP1 and muP3) shows a decrease in the luciferase reporter activity with SOX9 mutations. Firefly luciferase activity was measured after 48 h of transfection, and reporter activity was normalized to renilla luciferase activity (H , I) . Krt19 Cre /Cdk1 flox/flox / Tff1 −/− mice tissues were proceeded for immunofluorescence staining of CDK1, SOX9 and BCL X L. DAPI is used for nuclear staining (J , K) . scale bar = 20 μm

Article Snippet: IHC staining procedures were done by employing rabbit anti-human CDK1 antibody (ab133327, Abcam, Cambridge, MA) and SOX9 antibody (37447, Cell Signaling, Danvers, MA).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Control, Western Blot, Binding Assay, ChIP-qPCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK1 drives SOX9-mediated chemotherapeutic resistance in gastric cancer

doi: 10.1186/s13046-025-03523-3

Figure Lengend Snippet: Silencing CDK1 and SOX9 sensitizes AGS cells to CDDP treatment. AGS cells were transfected with SOX9, or empty vector (Ctrl) then treated with 20nM dinaciclib for 16 h. Whole cell lysate (WCL) was collected and subjected to western blot analysis, membranes were probed with CDK1, SOX9, BCL- X L, PARP, Cl-PARP and β-actin (A). WCL of AGS parental cells and CDDP-R cells (P1 and P2) were collected and subjected to western blot, membranes were probed with CDK1, SOX9, BCL- X L, and β-actin (B) . AGS parental cells and CDDP-R cells (P1, and P2) were transfected with siCtrl or siCDK1 for 72 h and IC50 was performed using ATP-GLO as instructed (C , D) . AGS parental cells and CDDP-R cells (P1 and P2) were transfected with siCDK1 or siCtrl for 72 h, WCL were collected and subjected to western blot. Membranes were probed with CDK1, p-DNMT1 (S154), SOX9, BCL- X L, and β-actin (E) . AGS parental cells and CDDP-R cells (P1, and P2) were transfected with siCtrl, siSOX9 for 72 h and IC50 was performed using ATP-GLO as instructed (F , G) . AGS parental cells and CDDP-R cells (P1 and P2) were transfected with siSOX9 or siCtrl for 72 h, WCL were collected and subjected to western blot analysis, membranes were probed with CDK1, SOX9, BCL- X L, and β-actin (H) . AGS parental cells and CDDP-R cells were treated with cisplatin and dinaciclib, either alone or in combination. Cell viability was then assessed using the ATP-Glo assay to determine the IC50 (I , K) . AGS parental cells and CDDP-R cells (P1) were treated with cisplatin 10 μm and dinaciclib 20nM, WCL was collected and subjected to western blot. Membranes were probed with CDK1, SOX9, BCL- X L, PARP, Cl-PARP, and β-actin (J , L)

Article Snippet: IHC staining procedures were done by employing rabbit anti-human CDK1 antibody (ab133327, Abcam, Cambridge, MA) and SOX9 antibody (37447, Cell Signaling, Danvers, MA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Glo Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK1 drives SOX9-mediated chemotherapeutic resistance in gastric cancer

doi: 10.1186/s13046-025-03523-3

Figure Lengend Snippet: CDK1 and SOX9 driving tumorigenesis in vitro and in vivo. A representative image of the tumoroids. Tumoroids were generated from patient-derived tumor xenografts (PDX498) then treated with Dinaciclib (10 nm), CDDP (10 μm) or combination for 72 h. scale bar = 100 μm. (A). A quantification of tumoroids size (B) and tumoroids number (C) for each condition, 5 random locations were measured and quantified using ImageJ. Tumoroids were collected and fixed then proceeded for immunofluorescence staining of CDK1 and SOX9 (D) , SOX9 and BCL- X L (E) . DAPI is used for nuclear staining and staining intensity was calculated using imageJ. scale bar = 20 μm. The tumor growth curve for PDX539 implanted in mice that are treated with Dinaciclib (20 mg/kg), CDDP (1 mg/kg), or combination (F) . Tumor width and length were measured two times weekly to calculate volume (G) . Kaplan-Meier survival curve (H) . PDX539 implanted in mice were treated with dinaciclib and cisplatin, tissues were collected and proceeded for immunohistochemistry (IHC) staining of Ki67 (I) and Cl-Caspase3 (K) . The staining intensity was calculated using imageJ ( J , L ). WCL was collected from the tissues and subjected to western blot analysis, membranes were probed with CDK1, SOX9 and BCL- X L, and β-actin (M) . A cartoon illustrating summary of the results is shown in panel (N)

Article Snippet: IHC staining procedures were done by employing rabbit anti-human CDK1 antibody (ab133327, Abcam, Cambridge, MA) and SOX9 antibody (37447, Cell Signaling, Danvers, MA).

Techniques: In Vitro, In Vivo, Generated, Derivative Assay, Immunofluorescence, Staining, Immunohistochemistry, Western Blot

Journal: Carcinogenesis

Article Title: Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells

doi: 10.1093/carcin/bgr017

Figure Lengend Snippet: 11p15.5 epigenetic and genetic states and expression of CDKN1C in breast cancer cells. (A) The DNA methylation status of the 11p15.5 ICR differentially methylated domain was analyzed by high-resolution bisulfite PCR pyrosequencing in primary tumors (n = 306), breast cancer cell lines (n = 52) and normal breast tissues (n = 19). Methylation levels of individual CpG sites (circles) are shown in a color gradient ranging from 0% (white) to 100% (blue). Left, diagram of CpG sites interrogated by the pyrosequencing assay. Right, results presented in rows of columns. (B) Multiple linear regression analysis of impact of 11p15.5 ICR methylation and genetic status on CDKN1C expression in breast cancer cells. The combined 11p15.5 ICR methylation, copy number and CDKN1C mRNA levels in 16 breast cancer cell lines were assessed by bisulfite PCR pyrosequencing, DNA copy number qPCR and qRT–PCR, respectively. DNA copy number of 11p15.5 ICR was quantified by absolute standard curve of cloned PCR products as described in supplementary Figure S4 (available at Carcinogenesis Online). Relative CDKN1C ΔCt expression values were obtained by normalizing to the 36B4 reference gene. (C) The effect of E2 on CDKN1C expression in breast cancer cells with 11p15.5 ICR hypermethylation. MDA-MB-453 and T47D cells were stimulated with 10 nM E2 or vehicle for 12 h. CDKN1C mRNA levels were assessed as described in Figure 1. Results are the average of two independent experiments. Columns, mean (n = 6); bars, SD.

Article Snippet: Approximately 300 ng of genomic DNA was bisulfite modified with EZ DNA Methylation Kit (Zymo Research, Irivine, CA) according to the manufacturer’s protocol.

Techniques: Expressing, DNA Methylation Assay, Methylation, Pyrosequencing Assay, Quantitative RT-PCR, Clone Assay

Journal: Carcinogenesis

Article Title: Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells

doi: 10.1093/carcin/bgr017

Figure Lengend Snippet: Potential mechanisms causing repression of CDKN1C in breast cancer cells. (A) Proposed model for epigenetic repression of CDKN1C through coordinated loop formation with the 11p15.5 ICR. CTCF binding to the ICR and CDKN1C locus and forms a long-range intrachromosomal loop via dimerization of CTCF. Ligand-bound ERα complex (orange and blue sphere) may mediate silencing through the formation of a secondary loop that serves both to sequester upstream tissue-specific enhancers and to recruit PRC2 and HDAC1 to the 11p15.5 ICR. CTCF serves as a scaffold to secure the PRC2 complex that methylates H3K27, leading to the formation of a repressive chromatin state at the CDKN1C locus. (B) Proposed regulatory mechanism of CDKN1C-AS. The formation of a double-stranded RNA may negatively regulate stability, transport and/or translation of the sense CDKN1C transcript. (C) Summary of three potential mechanisms causing CDKN1C repression in breast cancer cells. DNA methylation status of 11p15.5 ICR is indicated by large oval: white (unmethylated), black (methylated). Upper left, in the normal imprinted domain unmethylated 11p15.5 ICR on the paternal allele (♂) functions as a silencer and a promoter for KCNQ1OT1 transcription, repressing CDKN1C expression. The methylated maternal allele (♀) cannot function as a silencer or a promoter for KCNQ1OT1, thus permitting expression of CDKN1C. Upper right, the CDKN1C-AS transcript represses CDKN1C in trans, potentially through a double-stranded RNA mechanism. Under certain cellular conditions, this may be induced by estrogen-mediated upregulation of CDKN1C-AS. Lower left, DNA hypomethylation resulting from genetic loss of the methylated 11p15.5 ICR allele leads to aberrant domain silencer activity mediated by unrestricted CTCF binding and KCNQ1OT1 transcription, repressing CDKN1C expression. Lower right, estrogen induces KCNQ1OT1 transcription and CTCF recruitment to mediate ICR silencer activity, which in turn direct epigenetic repression of the CDKN1C locus.

Article Snippet: Approximately 300 ng of genomic DNA was bisulfite modified with EZ DNA Methylation Kit (Zymo Research, Irivine, CA) according to the manufacturer’s protocol.

Techniques: Binding Assay, DNA Methylation Assay, Methylation, Expressing, Activity Assay

Journal: Oncotarget

Article Title: TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection

doi: 10.18632/oncotarget.13130

Figure Lengend Snippet: A. Genome-wide gene expression levels were analyzed by RNA-seq. The diagram indicates the numbers of genes downregulated by <0.6-fold in three EBV-infected clones compared with MKN7_WT, i.e., 6,344 genes in MKN7_EB#1, 6,893 genes in MKN7_EB#2, and 6,312 genes in MKN7_EB#3. TET1 was included in the 3,735 genes that were downregulated in all the three clones, and TET2 was downregulated in MKN7_EB#1 and EB#3. B. Expression levels of TET genes were validated by real-time RT-PCR, and normalized against that of GAPDH . TET2 expression was markedly decreased in MKN7_EB#1, while TET1 expression was very low in both MKN7_WT and MKN7_EB#1. The experiment was done twice to confirm the similar result. C. Expression levels of TET genes were also analyzed in GES1, and normalized against that of GAPDH . All TET genes, especially TET2 , showed marked decreases in GES1_EBV compared with GES1_WT. The experiment was done twice to confirm the similar result. D, E. Immunoblotting analysis was conducted for TET2 and α-Tubulin in MKN7_WT and MKN7_EB#1 ( D ) and GES1_WT and GES1_EBV ( E ), and the analysis was done twice to confirm the similar result. The ratio of the intensity of TET2, measured by densitometer, to that of α-Tubulin was shown as a relative expression level.

Article Snippet: TET2 and the internal control α-Tubulin were detected by immunoblotting analysis using a rabbit anti-TET2 polyclonal antibody (1:1000, R1086-3, Abiocode, Agoura Hills, CA, USA) and a mouse anti-α-Tubulin monoclonal antibody (1:4000, sc-5286, Santa Cruz, Dallas, TX, USA).

Techniques: Genome Wide, Gene Expression, RNA Sequencing, Infection, Clone Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection

doi: 10.18632/oncotarget.13130

Figure Lengend Snippet: A. Expression level of TET2 was analyzed in MKN7 overexpressing EBV latent genes, LMP2A , ENBA1 , BARF0 and EBER1/2, and normalized against that of GAPDH . BARF0 and LMP2A significantly downregulated TET2 . B. Expression of 2,549 human miRNAs in MKN7_WT and the three EBV-infected clones were analyzed using a microarray. Among 87 miRNAs that were expected to target TET2 based on an in silico analysis, 15, 10, and 24 miRNAs were upregulated >1.3-fold in MKN7_EB#1, EB#2, and EB#3, respectively, and 7 miRNAs were upregulated in all the three clones. C. The 7 miRNAs were transfected into MKN7 and GES1, and real-time RT-PCR showed that TET2 expression levels decreased by 50-85% after 48 h. The experiment was done twice to confirm the similar result. D. Immunoblotting analysis was conducted for TET2 and α-Tubulin expression in GES1 transfected with the 7 miRNAs.

Article Snippet: TET2 and the internal control α-Tubulin were detected by immunoblotting analysis using a rabbit anti-TET2 polyclonal antibody (1:1000, R1086-3, Abiocode, Agoura Hills, CA, USA) and a mouse anti-α-Tubulin monoclonal antibody (1:4000, sc-5286, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Infection, Clone Assay, Microarray, In Silico, Transfection, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection

doi: 10.18632/oncotarget.13130

Figure Lengend Snippet: A. A TET2 -expressing vector was transfected into GES1 and the expression level of TET2 relative to GAPDH at 30 days after transfection was analyzed by real-time RT-PCR. Mock , GES1 cells transfected with an empty vector as negative controls. TET2OE , GES1 overexpressing TET2 . B. Immunoblotting analysis was conducted for TET2 and α-Tubulin expression in Mock and TET2OE cells. C. Representative results of hMeDIP-seq and MeDIP-seq around FRG1B are shown. The hydroxymethylation level of the region was increased in cells with TET2 overexpression, whereas the methylation level was increased in EBV infection. D. hMeDIP was repeated, and increase of hmC in 5′ region of FRG1B was validated by hMeDIP-PCR at the region indicated in Figure , and normalized against a positive control region NEDD9 . E. Increase of mC was validated by quantitative pyrosequencing assay at the region indicated in Figure . F. Among 2,619 hydroxymethylation target genes showing hydroxymethylation peaks within ±1 kb of the TSS in both Mock and TET2OE cells, 527 genes (20.1%) were methylation target genes during EBV infection ( P <1×10 -15 ). G. Among hydroxymethylation target genes in TET2OE, 1,231 genes showing increased hydroxymethylation from Mock to TET2OE were extracted as hydroxymethylation target genes by TET2. As many as 346 genes (28.1%) were methylation target genes during EBV infection ( P <1×10 -15 ). H. Methylation levels of hydroxymethylation target genes by TET2 were analyzed by Infinium, and average β values are shown. The 346 methylation target genes during EBV infection ( Group B ) showed marked increases of β values in GES1_EBV, while the other 885 genes that were not extracted as methylation target genes during EBV infection ( Group A ) showed slight, but still significant increases of β values in GES1_EBV. Both genes in Group A and Group B showed decreases of β values in TET2OE. WT , GES1_WT. EBV , GES1_EBV.

Article Snippet: TET2 and the internal control α-Tubulin were detected by immunoblotting analysis using a rabbit anti-TET2 polyclonal antibody (1:1000, R1086-3, Abiocode, Agoura Hills, CA, USA) and a mouse anti-α-Tubulin monoclonal antibody (1:4000, sc-5286, Santa Cruz, Dallas, TX, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Quantitative RT-PCR, Western Blot, Methylated DNA Immunoprecipitation, Over Expression, Methylation, Infection, Positive Control, Pyrosequencing Assay

Journal: Oncotarget

Article Title: TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection

doi: 10.18632/oncotarget.13130

Figure Lengend Snippet: A. Real-time RT-PCR showed a decrease in TET2 expression in shTET2 compared with shNON cells. The experiment was done twice to confirm the similar result. B. Immunoblotting analysis was conducted for TET2 and α-Tubulin expression in shNON and shTET2 cells. C. Among 13,150 genes that were unmethylated in shNON, none showed methylation in shTET2, i.e. , de novo methylation did not occur by TET2 knockdown alone. D. Three representative genes showed no methylation alterations in response to the knockdown of TET2 .

Article Snippet: TET2 and the internal control α-Tubulin were detected by immunoblotting analysis using a rabbit anti-TET2 polyclonal antibody (1:1000, R1086-3, Abiocode, Agoura Hills, CA, USA) and a mouse anti-α-Tubulin monoclonal antibody (1:4000, sc-5286, Santa Cruz, Dallas, TX, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Methylation, Knockdown

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 3: PPP2R2B Hypermethylation modulates MYC and P70 phosphorylation to Induce BEZ235 resistance. (A) PPP2R2B CpG island methylation was determined in CNE2 and CNE2/235 cell lines using a pyrosequencing assay. (B) The levels of PPP2R2B mRNA were determined using a quantitative PCR assay in parental and resistant cell lines. (C) The interactions between MYC and PP2A and between P70 and PP2A were detected by immunoprecipitation in CNE2 and CNE2/235 cell lines. (D) The levels of PPP2R2B mRNA were examined by q-PCR after transfection with PPP2R2B shRNA in CNE2 and HONE1. (E) Effect of PPP2R2B knockdown with shRNA on MYC and P70 phosphorylation in CNE2 cell line. (F) Effect of PPP2R2B knockdown on the interactions between MYC and PP2A and between P70 and PP2A in the CNE2 cell line based on immunoprecipitation. (G) The sensitivity of CNE2 to BEZ235 after PPP2R2B knockdown by PPP2R2B shRNA using the MTT assay.

Article Snippet: MTT was purchased from Sigma-Aldrich (St. Louis, Missouri), and phospho-AKT, phospho-MYC, MYC, GSK3α/β antibodies were from Santa Cruz Biotechnology (Indian Gulch, California).

Techniques: Phospho-proteomics, Methylation, Pyrosequencing Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Transfection, shRNA, Knockdown, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 4: Activation of PI3K/AKT/mTOR and PDK1/MYC survival pathway in BEZ235-resistant cells. (A) Effect of BEZ235 on the PI3K/AKT/mTOR and PDK1/MYC survival pathway in CNE2 and CNE2/235 cells by immunoblotting analysis. (B) Differential activation of the PI3K/AKT/mTOR and PDK1/MYC survival pathways in CNE2 and CNE2/235 cells by immunoblotting analysis. (C) Effect of PDK1 knockdown on P-AKT, P-P70, P-Myc, and PIM1 expression in CNE2/235 cells. (D) Sensitivity to BEZ235 in CNE2/235 cells after PDK1 knockdown using the MTT assay. (E) Effect of MYC knockdown on the sensitivity to BEZ235 in CNE2/235 cells. (F) Effect of P70(S6K) knockdown on the sensitivity to BEZ235 in CNE2/235 cells. (G) Effect of the PDK1 inhibitor GSK2334470 on MYC and P70 phosphorylation in CNE2/235 cell line by immunoblotting analysis. (H) Sensitivity to GSK2334470 in CNE2/235 cells after treatment with GSK2334470 for 5 d using the MTT assay. (I) Inhibitory effect of GSK2334470 in combination with BEZ235 on cell proliferation in CNE2/235 cells using the MTT assay. (J) IC50 values of BEZ235 with or without GSK2334470 in CNE2 and CNE2/235 cell lines. The data shown are representative of 3 individual experiments.

Article Snippet: MTT was purchased from Sigma-Aldrich (St. Louis, Missouri), and phospho-AKT, phospho-MYC, MYC, GSK3α/β antibodies were from Santa Cruz Biotechnology (Indian Gulch, California).

Techniques: Activation Assay, Western Blot, Knockdown, Expressing, MTT Assay, Phospho-proteomics

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 5: PIM1 Induces MYC Phosphorylation that Drivers BEZ235 Resistance. (A) Differential expression of PIM1, P-PLK, and PLK proteins in the parental and BEZ235-resistant cells by immunoblotting analysis. (B) The interaction between MYC and PIM1 was detected using an immunoprecipitation assay in parental and resistant cell lines. (C) Effect of PIM1 knockdown on P-Myc expression in CNE2/235 and HONE1/235 cells. (D) Sensitivity to BEZ235 in BEZ235-resistant cells after PIM1 knockdown using the MTT assay. (E) Effect of the MYC inhibitor JQ-1 on MYC phosphorylation in BEZ235-resistant cell lines. (F) Sensitivity of BEZ235-resistant cells to JQ-1 using the MTT assay.

Article Snippet: MTT was purchased from Sigma-Aldrich (St. Louis, Missouri), and phospho-AKT, phospho-MYC, MYC, GSK3α/β antibodies were from Santa Cruz Biotechnology (Indian Gulch, California).

Techniques: Phospho-proteomics, Quantitative Proteomics, Western Blot, Immunoprecipitation, Knockdown, Expressing, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 6: Inhibition of DNA Methyltransferase activity reverses BEZ235 resistance. (A) The mRNA levels of DNA methyltransferases were analyzed in CNE2 and CNE2/235 cell lines by q-PCR. (B) PPP2R2B and PTEN mRNA levels were analyzed in resistant cell lines after treatment with the DNA methylation inhibitor decitabine at the indicated concentrations for 72 h. (C) Effects of decitabine on the expression of survival pathway proteins at the indicated concentrations. (D) Effects of decitabine on the binding of MYC and PP2A and of P70 and PP2A in the CNE2/235 cell line. Cells were treated with 1 μM decitabine for 48 h. Immunoprecipitation was conducted. (E) Effect of the DNA methylation inhibitor decitabine on cell proliferation in the parental and resistant cell lines using the MTT assay. (F) Inhibitory effect of decitabine and BEZ235 on cell proliferation in CNE2 and CNE2/235 cell lines. Cells were treated for 5 d at the indicated concentrations. The MTT assay was conducted. (G) The IC50 values of BEZ235 with or without decitabine were determined in CNE2 and CNE2/235 cell lines using the MTT assay. The data shown are representative of 3 individual experiments.

Article Snippet: MTT was purchased from Sigma-Aldrich (St. Louis, Missouri), and phospho-AKT, phospho-MYC, MYC, GSK3α/β antibodies were from Santa Cruz Biotechnology (Indian Gulch, California).

Techniques: Inhibition, Activity Assay, DNA Methylation Assay, Expressing, Binding Assay, Immunoprecipitation, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 1: DNA hypermethylation in acquired dual PI3K/mTOR inhibitors resistant cells. (A) Inhibitory effects of BEZ235 on CNE2 and HONE1 cell proliferation. Cell growth was assessed using the MTT assay after treatment with BEZ235 for 5 d. (B) Effect of BEZ235 on cell proliferation in the parental nasopharyngeal carcinoma cell lines and their corresponding acquired BEZ235 sublines using the MTT assay. (C) Effect of GDC0980 on cell proliferation in the parental nasopharyngeal carcinoma cell lines and their corresponding acquired BEZ235 sublines using the MTT assay. ( D) IC50 values of BEZ235 in parental cell lines and their corresponding resistant cells. The data shown are representative of 3 individual experiments. (E) Representative microscopic images of the parental cell lines CNE2 and HONE1 and their resistant CNE2/235 and HONE1/235 cells grown in 6-well plates. (F) Growth curves were calculated for 7 d using the MTT assay with or without 0.4 μM BEZ235. (G) The cell cycle was analyzed in parental cell lines and resistant cell lines by PI staining and analyzed by flow cytometry. (H) Parental cells and their resistant counterparts were plated in matrigel-coated 96-well plates. Adhesion was analyzed using the MTT assay. (*p < 0.05; **p < 0.01). (I) Gene methylation was determined using Illumina Methylation BeasChip assays in the CNE2 and CNE2/235 cell lines. Dots on the bit line indicate no difference between CNE2 and CNE2/235.

Article Snippet: BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals.

Techniques: MTT Assay, Staining, Flow Cytometry, Methylation

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 2: PTEN hypermethylation Activates the PI3K/mTOR Signaling pathway to induce BEZ235 resistance. (A) PTEN CpG island methylation was determined by pyrosequencing assays in CNE2 and CNE2/235 cell lines. (B) PTEN mRNA levels were examined using a quantitative PCR assay in parental and resistant cell lines. (C) Effect of PTEN knockdown by siRNA on AKT, GSK3β and P70 phosphorylation in CNE2 and HONE1 cell lines by immunoblotting analysis. (D) Sensitivity to BEZ235 after PTEN knockdown by siRNA in CNE2 and HONE1 cell lines.

Article Snippet: BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals.

Techniques: Methylation, Real-time Polymerase Chain Reaction, Knockdown, Phospho-proteomics, Western Blot

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 3: PPP2R2B Hypermethylation modulates MYC and P70 phosphorylation to Induce BEZ235 resistance. (A) PPP2R2B CpG island methylation was determined in CNE2 and CNE2/235 cell lines using a pyrosequencing assay. (B) The levels of PPP2R2B mRNA were determined using a quantitative PCR assay in parental and resistant cell lines. (C) The interactions between MYC and PP2A and between P70 and PP2A were detected by immunoprecipitation in CNE2 and CNE2/235 cell lines. (D) The levels of PPP2R2B mRNA were examined by q-PCR after transfection with PPP2R2B shRNA in CNE2 and HONE1. (E) Effect of PPP2R2B knockdown with shRNA on MYC and P70 phosphorylation in CNE2 cell line. (F) Effect of PPP2R2B knockdown on the interactions between MYC and PP2A and between P70 and PP2A in the CNE2 cell line based on immunoprecipitation. (G) The sensitivity of CNE2 to BEZ235 after PPP2R2B knockdown by PPP2R2B shRNA using the MTT assay.

Article Snippet: BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Methylation, Pyrosequencing Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, Transfection, shRNA, Knockdown, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 4: Activation of PI3K/AKT/mTOR and PDK1/MYC survival pathway in BEZ235-resistant cells. (A) Effect of BEZ235 on the PI3K/AKT/mTOR and PDK1/MYC survival pathway in CNE2 and CNE2/235 cells by immunoblotting analysis. (B) Differential activation of the PI3K/AKT/mTOR and PDK1/MYC survival pathways in CNE2 and CNE2/235 cells by immunoblotting analysis. (C) Effect of PDK1 knockdown on P-AKT, P-P70, P-Myc, and PIM1 expression in CNE2/235 cells. (D) Sensitivity to BEZ235 in CNE2/235 cells after PDK1 knockdown using the MTT assay. (E) Effect of MYC knockdown on the sensitivity to BEZ235 in CNE2/235 cells. (F) Effect of P70(S6K) knockdown on the sensitivity to BEZ235 in CNE2/235 cells. (G) Effect of the PDK1 inhibitor GSK2334470 on MYC and P70 phosphorylation in CNE2/235 cell line by immunoblotting analysis. (H) Sensitivity to GSK2334470 in CNE2/235 cells after treatment with GSK2334470 for 5 d using the MTT assay. (I) Inhibitory effect of GSK2334470 in combination with BEZ235 on cell proliferation in CNE2/235 cells using the MTT assay. (J) IC50 values of BEZ235 with or without GSK2334470 in CNE2 and CNE2/235 cell lines. The data shown are representative of 3 individual experiments.

Article Snippet: BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals.

Techniques: Activation Assay, Western Blot, Knockdown, Expressing, MTT Assay, Phospho-proteomics

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 5: PIM1 Induces MYC Phosphorylation that Drivers BEZ235 Resistance. (A) Differential expression of PIM1, P-PLK, and PLK proteins in the parental and BEZ235-resistant cells by immunoblotting analysis. (B) The interaction between MYC and PIM1 was detected using an immunoprecipitation assay in parental and resistant cell lines. (C) Effect of PIM1 knockdown on P-Myc expression in CNE2/235 and HONE1/235 cells. (D) Sensitivity to BEZ235 in BEZ235-resistant cells after PIM1 knockdown using the MTT assay. (E) Effect of the MYC inhibitor JQ-1 on MYC phosphorylation in BEZ235-resistant cell lines. (F) Sensitivity of BEZ235-resistant cells to JQ-1 using the MTT assay.

Article Snippet: BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals.

Techniques: Phospho-proteomics, Quantitative Proteomics, Western Blot, Immunoprecipitation, Knockdown, Expressing, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 6: Inhibition of DNA Methyltransferase activity reverses BEZ235 resistance. (A) The mRNA levels of DNA methyltransferases were analyzed in CNE2 and CNE2/235 cell lines by q-PCR. (B) PPP2R2B and PTEN mRNA levels were analyzed in resistant cell lines after treatment with the DNA methylation inhibitor decitabine at the indicated concentrations for 72 h. (C) Effects of decitabine on the expression of survival pathway proteins at the indicated concentrations. (D) Effects of decitabine on the binding of MYC and PP2A and of P70 and PP2A in the CNE2/235 cell line. Cells were treated with 1 μM decitabine for 48 h. Immunoprecipitation was conducted. (E) Effect of the DNA methylation inhibitor decitabine on cell proliferation in the parental and resistant cell lines using the MTT assay. (F) Inhibitory effect of decitabine and BEZ235 on cell proliferation in CNE2 and CNE2/235 cell lines. Cells were treated for 5 d at the indicated concentrations. The MTT assay was conducted. (G) The IC50 values of BEZ235 with or without decitabine were determined in CNE2 and CNE2/235 cell lines using the MTT assay. The data shown are representative of 3 individual experiments.

Article Snippet: BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals.

Techniques: Inhibition, Activity Assay, DNA Methylation Assay, Expressing, Binding Assay, Immunoprecipitation, MTT Assay

Journal: Oncotarget

Article Title: Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors.

doi: 10.18632/oncotarget.3016

Figure Lengend Snippet: Figure 7: Targeting methyltransferase overcomes BEZ235 resistance in vivo. (A) (B) Xenograft tumor growth of CNE2 and CNE2/235 cells in nude mice treated with BEZ235 at 25 mg/kg per day or decitabine 1.5 mg/kg three times per week. (p values in CNE2: p = 0.27 in Decitabine vs slovent, p < 0.01 in BEZ235 vs NS, p < 0.01 in combination vs solvent, p = 0.55 in combination vs BEZ235; p values in CNE2/235: p = 0.90 in Decitabine vs slovent, p = 0.03 in BEZ235 vs NS, p < 0.01 in combination vs solvent, p = 0.03 in combination vs Decitabine) (C) The inhibitory rates of tumor growth were calculated as 100% × (1-average treated tumor size/ average control tumor size). (D) Antitumor efficacy of BEZ235 and decitabine in vivo by tumor weight. Nude mice were killed, and tumors were isolated to weigh each tumor. (E) The mRNA levels of PPP2R2B and PTEN were determined by q-PCR in parental and resistant xenografts. (F) Protein expression of survival pathway components was determined by immunoblotting analysis in parental and resistant xenografts. (G) Protein expression of survival pathway components was determined in parental and resistant xenografts by immunoblotting analysis following treatment with decitabine in vivo.

Article Snippet: BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals.

Techniques: In Vivo, Solvent, Control, Isolation, Expressing, Western Blot