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: Clinical Epigenetics

Article Title: A randomized controlled trial of folic acid intervention in pregnancy highlights a putative methylation-regulated control element at ZFP57

doi: 10.1186/s13148-019-0618-0

Figure Lengend Snippet: ZFP57 upstream region is a methylation-dependent regulator of transcription at this locus. a Schematic as in Fig. above but showing difference in methylation (Δ β ) between HCT116 WT cells vs HCT116 DKO cells. The intron/exon structure and positions of the forward (FW) and reverse (RV) primers for RT-(q)PCR on the ZFP57 gene are also shown. b Methylation levels at individual CpG covered by the pyrosequencing assay in WT (HCT116) and knockout (DKO) cells. Values are shown as mean +/− SD for each site: * p < 0.05; ** p < 0.01; *** p < 0.001. c RT-PCR showing upregulation using the primers indicated in a , key as above. CTRL, positive control (human reference total RNA); NTC, negative control (no template control); 100 bp, size standards ladder; ACTB , β-actin loading control. d Confirmation of upregulation by RT-qPCR using the same primers, values normalized to HPRT ; FC, fold change. e Methylation levels using pyroassay as in B but in 5-aza-dC treated SH-SY5Y cells (5-aza-dC), as compared to untreated (UT). f RT-PCR for 5-aza-dC treated cells from e . g RT-qPCR confirmation of ZFP57 upregulation in 5-aza-dC-treated SH-SY5Y cells

Article Snippet: Human reference total RNA was used as a positive control for expression (Clontech, UK).

Techniques: Methylation, Pyrosequencing Assay, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Quantitative RT-PCR

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: 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: Nucleic Acids Research

Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

doi: 10.1093/nar/gkw159

Figure Lengend Snippet: ( A ) Schematic representation of the dCas9-DNMT3A fusion protein in complex with sgRNA and its target DNA sequence. The sgRNA is bound in a cleft between the recognition lobe (RecI, II and III domains) and the nuclease lobe (HNH, RuvC and PI domains) of Cas9 protein. The C–terminus of Cas9 is located on the PAM–interacting (PI) domain and faces the side where the bound genomic DNA protrudes with its 3′ end relative to the sgRNA sequence. The sgRNA is a synthetic fusion between bacterial crRNA and tracrRNA, with guide sequence and tracrRNA part shown in different colors. The catalytic domain of DNMT3A recruits its partner for dimerization and DNMT3L proteins in vivo (dashed lightened symbols). NLS, nuclear localization signal; GS, Gly 4 Ser peptide linker. ( B ) Domain structure of the dCas9–DNMT3A fusion protein. The nuclease-inactivating mutations D10A and H840A of Streptococcus pyogenes Cas9 are indicated. Deactivated Cas9 was fused to the catalytic domain of the human de novo DNA methyltransferase 3A (DNMT3A CD) using a short Gly 4 Ser peptide (GS). The dCas9–DNMT3A is expressed as a bicistronic mRNA, along with puromycin resistance gene (PuroR, shown) or EGFP gene, thus enabling selection of transfected cells. The PuroR (or EGFP) moiety is separated during translation by action of the T2A self-cleaving peptide. The inactive fusion methyltransferase (dCas9-DNMT3A-ANV) for use as a negative control contains an additional substitution (E155A*) in the active site of DNMT3A. 3x FLAG, epitope tag; NLS, nuclear localization signal.

Article Snippet: Undesired BbsI restriction site within the catalytic domain of human DNMT3A from the plasmid pcDNA3/Myc-DNMT3A (Addgene plasmid # 35521, a gift from Arthur Riggs) ( ) was removed by site-directed mutagenesis.

Techniques: Sequencing, In Vivo, Selection, Transfection, Negative Control, FLAG-tag

Journal: Nucleic Acids Research

Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

doi: 10.1093/nar/gkw159

Figure Lengend Snippet: Targeting the dCas9-DNMT3A tool to the BACH2 and IL6ST regulatory regions. The human ( A ) BACH2 and ( B ) IL6ST loci are shown with positions of the pyrosequencing assays (BACH2-A assay, BACH2-B assay and IL6ST-A assay) indicated by blue rectangles. Magnified insets show individual CpG sites analyzed by pyrosequencing, with arrows (aligned to the magnified regions) indicating 20 bp binding sites of sgRNAs used to guide the dCas9-DNMT3A construct. Arrows point toward the PAM sequence.

Article Snippet: Undesired BbsI restriction site within the catalytic domain of human DNMT3A from the plasmid pcDNA3/Myc-DNMT3A (Addgene plasmid # 35521, a gift from Arthur Riggs) ( ) was removed by site-directed mutagenesis.

Techniques: Binding Assay, Construct, Sequencing

Journal: Nucleic Acids Research

Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

doi: 10.1093/nar/gkw159

Figure Lengend Snippet: ( A ) Activity of the dCas9-DNMT3A tool guided by sgRNA8 or sgRNA3 was quantified using the BACH2-A and BACH2-B assays, respectively. The graphs show increase in CpG methylation level relative to the mock–transfected cells. Increase in the methylation level for active constructs could be observed some distance downstream from the PAM sequence (e.g. sgRNA8) or at both sides of the binding site (e.g. sgRNA3) but not at the binding site itself (shaded region indicated with arrows labeled with sgRNA names). Inactive (dCas9–DNMT3A–ANV) or non-targeting (sgRNA with no binding site in the human genome) did not show any significant increase in methylation. ( B ) Summary profile of the dCas9–DNMT3A activity is shown as absolute methylation fraction increase (compared to mock–transfected cells) relative to the distance of a CpG site from the PAM sequence. The summary activity profile is based on the activity of all sgRNAs targeting the BACH2 promoter and the IL6ST promoter. Vertical solid red lines represent the binding region complementary to the sgRNA. Results of all experiments were integrated by orienting the sgRNAs in the same direction and aligning the PAM sequence to position zero. Different relative positions of binding sites and pyrosequencing assays enabled construction of an activity profile covering a wide region when all available experimental data were used. The peak of CpG methylation activity extends over about 25–30 nucleotide pairs centered at the 27th nucleotide (vertical dashed purple line) downstream from the targeted PAM sequence. Another much smaller peak was consistently observed at the approximately same distance upstream from the sgRNA binding site. Gray bars represent the CpG methylation level increase observed within a single experiment (error bars show standard deviation). Each gray bar summarizes data for one CpG site position and also serves as a visual guide showing the density of coverage with experimental points. The blue curve shows LOESS smoothing of the data from multiple experiments. The brown dotted curve shows smoothed data for CpG methylation level increase by the inactive construct.

Article Snippet: Undesired BbsI restriction site within the catalytic domain of human DNMT3A from the plasmid pcDNA3/Myc-DNMT3A (Addgene plasmid # 35521, a gift from Arthur Riggs) ( ) was removed by site-directed mutagenesis.

Techniques: Activity Assay, CpG Methylation Assay, Transfection, Methylation, Construct, Sequencing, Binding Assay, Labeling, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

doi: 10.1093/nar/gkw159

Figure Lengend Snippet: Targeted CpG methylation and transcriptional silencing of the IL6ST gene by the dCas9–DNMT3A tool. ( A ) Increase in CpG methylation level relative to the mock–transfected cells in the IL6ST promoter region targeted by either individual sgRNAs (1–4) or by pooled sgRNAs 1–4. ( B ) Expression level of the IL6ST gene as measured using RT–qPCR revealed a statistically significant decrease ( P < 0.05) in the transcript level following transfection with pooled sgRNAs 1–4. Fold change is relative to mock-transfected cells. Error bars represent standard deviation. Non-targeting sgRNA served as negative control.

Article Snippet: Undesired BbsI restriction site within the catalytic domain of human DNMT3A from the plasmid pcDNA3/Myc-DNMT3A (Addgene plasmid # 35521, a gift from Arthur Riggs) ( ) was removed by site-directed mutagenesis.

Techniques: CpG Methylation Assay, Transfection, Expressing, Quantitative RT-PCR, Standard Deviation, Negative Control

Journal: Nucleic Acids Research

Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

doi: 10.1093/nar/gkw159

Figure Lengend Snippet: Targeted CpG methylation and transcriptional silencing of the BACH2 gene using the dCas9–DNMT3A tool. ( A ) CpG methylation level increased relative to mock-transfected cells in the BACH2 promoter region when targeted by either individual sgRNAs (6–8) or pooled sgRNAs 1–8. The region is covered by the pyrosequencing assay BACH2–A. Note that sgRNAs 6–8 bind close to the region covered by the BACH2–A assay, while sgRNAs 1–5 bind further downstream (see also Figure for reference). ( B ) CpG methylation level increased relative to mock-transfected cells in the BACH2 promoter region (covered by the pyrosequencing assay BACH2–B) when targeted by either individual sgRNAs (1–5) or pooled sgRNAs 1–8. Note that sgRNAs 1–5 bind close to the region covered by the BACH2–B assay, while sgRNAs 6–8 bind further upstream (see also Figure for reference). ( C ) Expression level of the BACH2 gene as measured using RT–qPCR revealed a statistically significant ( P < 0.05) decrease in the transcript level following transfection with dCas9–DNMT3A and pooled sgRNAs 1–8. A lower but statistically significant decrease in expression was observed with matching pooled sgRNAs 1–8 used with inactive dCas9–DNMT3A constructs, which is consistent with CRISPR interference. Fold change is relative to mock-transfected cells. Error bars represent standard deviation. Non-targeting sgRNA served as negative control.

Article Snippet: Undesired BbsI restriction site within the catalytic domain of human DNMT3A from the plasmid pcDNA3/Myc-DNMT3A (Addgene plasmid # 35521, a gift from Arthur Riggs) ( ) was removed by site-directed mutagenesis.

Techniques: CpG Methylation Assay, Transfection, Pyrosequencing Assay, Expressing, Quantitative RT-PCR, Construct, CRISPR, Standard Deviation, Negative Control

Journal: Nucleic Acids Research

Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

doi: 10.1093/nar/gkw159

Figure Lengend Snippet: ( A ) Time-course evaluation of targeted CpG methylation in cells transfected with active or inactive dCas9-DNMT3A construct co-expressed with individual targeting sgRNAs. CpG methylation level increase was measured at different time points after transfection over a time period of 40 days. We analyzed CpG sites falling within the peak of methylation activity 18–42 bp downstream from the PAM sequence (positions of CpG sites relative to the PAM sequence are given in brackets). The time point when DNA methylation reaches its maximum is marked with a purple dashed vertical line. Left panel: Methylation of the BACH2-A fragment in cells co-expressing BACH2-sgRNA8. Right panel: Methylation of the IL6ST-A fragment in cells co-expressing IL6ST-sgRNA3. ( B ) Relative amount of plasmid DNA per cell, as determined by qPCR, decreases sharply in the 10 days following transfection. ( C ) Even without puromycin selection (using the construct co–expressing EGFP instead of puromycin resistance gene), the detected expression of the Cas9-DNMT3A construct (measured via co-expression of EGFP) decreases within 10 days after transfection. Arbitrary units (AU) represent fluorescence intensity per field of view normalized to the total number of cells counted under visible light.

Article Snippet: Undesired BbsI restriction site within the catalytic domain of human DNMT3A from the plasmid pcDNA3/Myc-DNMT3A (Addgene plasmid # 35521, a gift from Arthur Riggs) ( ) was removed by site-directed mutagenesis.

Techniques: CpG Methylation Assay, Transfection, Construct, Methylation, Activity Assay, Sequencing, DNA Methylation Assay, Expressing, Plasmid Preparation, Selection, Fluorescence

Journal: PLoS Genetics

Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

doi: 10.1371/journal.pgen.1007723

Figure Lengend Snippet: (A) Schematic of CFTR-Expression Minigene with full-length introns 25 and 26 (EMG-i25-i26) constructed in pcDNA5FRT plasmid. CFTR expression is driven by a CMV promoter. The location of each studied variant is shown relative to CFTR exons and regions predicted to elicit NMD. (B) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with nonsense or frameshift variants, as indicated. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Immunoblot (IB) of the steady state amounts of immature core-glycosylated ( band B ) and the mature complex-glycosylated mature CFTR protein ( band C ). Lysates were collected from HEK293 cells expressing WT-EMG or EMGs with different PTC-generating variants. Lysates from cells expressing either intronless WT CFTR or F508del served as controls, or empty vector as negative control. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody (596 # Cystic Fibrosis Foundation Therapeutics). (D) Schematic illustration showing three groups of 3’- nonsense variants based on mRNA stability and protein maturity. (E) Immunoblot of HEK293 stable cells expressing Q1390X or E1418X. The cells were incubated for 48 h with DMSO (.03%) or corrector compounds (lumacaftor and tezacaftor either alone or in combination—3 μM each). CFTR was visualized with anti-CFTR antibody, 596 (CFFT). (F) A representative Ussing chamber tracing of EMG E1418X-expressing CFBE stable cells grown on snap-wells. Short-circuit current (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. Cells were mounted on Ussing chambers to measure CFTR mediated chloride channel activity as a proxy of CFTR function. After stabilization of the basal current, forskolin (10 μM) was added to the basolateral chamber followed by potentiator, ivacaftor (10 μM), and CFTR Inhibitor 172 (10 μM) added to the apical chambers. Inh-172 was added earlier in DMSO no ivacaftor (dashed blue line) treated cells. (G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i25-i26. P value was determined by one way ANOVA. **** P ≤0.0001, ** P ≤0.01, * P ≤0.05, and n.s. (not significant, P >0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

Techniques: Expressing, Construct, Plasmid Preparation, Variant Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control, Incubation, Activity Assay

Journal: PLoS Genetics

Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

doi: 10.1371/journal.pgen.1007723

Figure Lengend Snippet: (A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. ( G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated I sc without modulator treatment in cells expressing EMG i21-i22. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001), n.s. (not significant, P >0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

Techniques: Expressing, Construct, Plasmid Preparation, Variant Assay, Quantitative RT-PCR, Residue, Transfection, Stable Transfection

Journal: PLoS Genetics

Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

doi: 10.1371/journal.pgen.1007723

Figure Lengend Snippet: (A) Relative expression of the alternate CFTR allele in the primary nasal cells of CF individuals carrying exon 22 nonsense variant. Pyrosequencing assay was designed such that exon 22 with upstream and downstream flanking exons was amplified from the corresponding cDNA preparations. Sequencing primer yielded relative abundances of alternate alleles at the respective loci where nucleotide change occurred. (B ) CFTR mRNA decay in HEK293 cells stably expressing wild type EMG or EMG harboring nonsense variants R1158X or S1196X. Actinomycin D (3 μg/ml) was added at time 0 to induce transcriptional shut-down. Cells were collected at the indicated time points. Levels of the CFTR mRNAs were assessed by RT-qPCR, normalized to B2M mRNA and displayed as a percentage of the levels at t = 0. Mean ± SEM ( n = 3) (C) Efficiency of siRNA mediated knock down of UPF1 detected on IB of whole cell lysates collected from HEK293 cells stably expressing either R1158X or S1196X. GAPDH siRNA and non-target (NT) siRNA were used as positive and negative controls respectively. Beta-Actin was used as loading control. (D) Effect of direct NMD inhibition on the level of CFTR transcript by siRNA mediated knock down of UPF1 in HEK293 cells stably expressing either R1158X or S1196X. Levels of the CFTR mRNAs were assessed by RT-qPCR and normalized to B2M mRNA. Mean ± SEM, n = 3 independent biological triplicates, P value was determined by two way ANOVA. ** ( P ≤0.01) and *** ( P ≤0.001) indicate significant difference when compared with CFTR mRNA abundance in untreated cells (E) Short-circuit (I sc ) tracings of CFBE-S1196X stable cells recorded in Ussing chambers after direct inhibition of NMD by UPF1 . Cells were transfected with Upf1 siRNA at 50% confluency for 4 days before being mounted on Ussing chambers. GAPDH and non-targeted (NT) siRNA transfections were used as controls. Cells were incubated with lumacaftor (3 μM) or DMSO (0.03%) during last 48h of siRNA transfections. (F) Stacked bar graphs indicate effect of UPF1 siRNA in combination with CFTR modulators. Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3). P value was determined by one way ANOVA. ** ( P ≤0.01) indicate significant difference when compared with forskolin stimulated CFTR function in NT siRNA transfected cells and **** ( P ≤0.0001) indicate significant difference when compared with ivacaftor activated CFTR function in NT siRNA transfected cells incubated with or without lumacaftor. WT-CFTR function represents forskolin stimulated ΔI sc without modulator treatment in cells expressing EMG i21-i22.

Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

Techniques: Expressing, Variant Assay, Pyrosequencing Assay, Amplification, Sequencing, Stable Transfection, Quantitative RT-PCR, Knockdown, Control, Inhibition, Transfection, Incubation

Journal: PLoS Genetics

Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

doi: 10.1371/journal.pgen.1007723

Figure Lengend Snippet: (A) A schematic illustration of CFTR-Expression Minigene with introns 14–18 (introns 14 and 16 are full-length, and 15, 17, & 18 are abridged). Arrow indicates location of E831X (top). CFTR mRNA splicing patterns of the total RNA extracted from HEK293 cells transiently transfected with E831X-EMG. (B) Steady state amounts of different isoforms of CFTR produced from E831X-EMG-i14-i18 expressed transiently in HEK293 cells. Lysates from cells expressing WT-EMG i14-i18, intronless WT CFTR or F508del served as positive controls, and empty vector as negative control. Immunoblot (IB) was probed with anti-CFTR antibody, 596 (CFFT). Horizontal arrows indicate to isoforms corresponding to (i) a normal codon (E831) substituted with a stop codon, (ii) deletion of complete exon 16, and (iii) deletion of a single amino acid E831. Beta-Actin was used as loading control. (C) Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing E831X mounted on Ussing chamber. Cells were treated for 48 h with correctors (lumacaftor/tezacaftor or both, 3 μM each) and acutely with potentiator (ivacaftor, 10 μM). Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean±SEM (n = 3). P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i14-i18. (D ) A tracing of CFTR function observed in primary nasal epithelial cells of an individual harboring E831X/F508del. CF-Human nasal epithelial (HNE) cells were treated for 24 h with lumacaftor and tezacaftor, 3 μM each, and acutely with Ivacaftor (10 μM). Stacked bar graph is a comparison of improvement in CFTR function of E831X/F508del vs F508del/Indel. Alternate Indel alleles were either 2184insA, 2183delAA>G, or 3659del C. P value was determined by one way ANOVA. ** indicates significant difference ( P ≤0.01) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated E831X/F508del HNEs. * ( P ≤0.05) when compared with CFTR function in modulator treated F508del/Indel HNEs.

Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

Techniques: Expressing, Transfection, Produced, Plasmid Preparation, Negative Control, Western Blot, Control, Comparison

Journal: PLoS Genetics

Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

doi: 10.1371/journal.pgen.1007723

Figure Lengend Snippet: (A) Left panel—Sanger sequencing, and right panel—fragment analysis. Total RNA was extracted from the conditionally reprogrammed nasal epithelial cells of CF individual with F508del/L88X genotype. RT-PCRs were performed using CFTR-specific primers to amplify L88X and F508del regions. Area under the peak was used to determine expression of L88X transcript compared to F508del. (B) RNA-seq of the primary human nasal epithelial cells of healthy and L88X/F508del individuals. Density profile of all expressed genes (top), and relative transcript counts of L88X compared to F508del (bottom). (C) Schematic of CFTR-Expression Minigene with abridged introns 1, 2, 3, 4 and 5 constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (D) Graph shows relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M. Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. No significant difference (n.s.) ( P >0.01) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (E) Immunoblot of the naturally occurring 5’-truncations on the steady state amounts of CFTR protein expressed in HEK293 cells. CFTR was visualized with anti-CFTR antibody-596 (CFFT), and anti-Na + K + ATPase served as control. (F) CFTR function measured in CFBE stable expressing L88X. Cells were incubated for 24 h with readthrough compound (G418, 5 μM and 25 μM)/ corrector (lumacaftor, 3 μM) or both. Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing L88X mounted on Ussing chamber. Change in Isc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean ± SEM (n = 3). P value was determined by one way ANOVA. **** ( P ≤0.0001), and *** ( P ≤0.001) indicate significant difference when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i1-i5.

Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

Techniques: Sequencing, Expressing, RNA Sequencing, Construct, Plasmid Preparation, Variant Assay, Residue, Western Blot, Control, Incubation

Journal: Oncotarget

Article Title: Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer

doi: 10.18632/oncotarget.17527

Figure Lengend Snippet: ( A ) qRT-PCR analysis of GPX7 gene expression in 7 gastric cancer cell lines and a normal gastric mucosa sample, showing undetectable GPX7 mRNA in all 7 gastric cancer cell lines examined. ( B ) Western blotting analysis of GPX7 protein in the 7 gastric cancer cell lines. ( C ) A schematic drawing shows a CpG island in GPX7 gene promoter, and pyrosequencing assay location. Each vertical bar represents a CpG site. TSS, transcription start site. DNA methylation level of 8 CpG sites in the GPX7 promoter was quantitated by pyrosequencing. ( D ) and ( E ) show representative pyrosequencing profiles of AGS and a normal gastric mucosa sample respectively. ( F ) Displays DNA methylation level of GPX7 promoter in the 7 gastric cancer cell lines, showing more than 50% methylation level in all the cell lines.

Article Snippet: The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, Proteintech Group, Chicago, IL USA), and anti-PARP and cleaved PARP (Cell Signaling).

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Pyrosequencing Assay, DNA Methylation Assay, Methylation

Journal: Oncotarget

Article Title: Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer

doi: 10.18632/oncotarget.17527

Figure Lengend Snippet: ( A ) Downregulation of the GPX7 gene expression was found in 48.8% primary gastric cancer samples as compare to their matched normal samples from the same patients. ( B ) A schematic profile shows GPX7 methylation of the 8 CpG sites in 2 representative matched normal (NG) and tumor (T) samples. ( C ) Shows the average DNA methylation level of GPX7 promoter in 45 normal and 45 tumor samples. *** p < 0.001. ( D ) The DNA methylation level change of GPX7 promoter in 45 individual matched normal and tumor samples from the same patients is indicated ( p < 0.001). ( E ) The Spearman rank correlation analysis between GPX7 promoter methylation and gene expression in all the samples, displays a reverse correlation between DNA methylation and gene expression ( r = −0.27, p = 0.01). ( F ) and ( G ) show GPX7 gene expression and promoter methylation after 5-aza and/or TSA treatment in AGS (F) and SNU1 (G) cells. Relative GPX7 expression folds are shown in the right panels. DNA methylation levels of corresponding samples are shown on the left panels. 5-Aza, 5-Aza-2′ deoxycytidine. TSA, Trichostatin-A. DMSO, Dimethyl Sulfoxide. * p < 0.05, ** p < 0.01, *** p < 0.001, as compared to DMSO control.

Article Snippet: The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, Proteintech Group, Chicago, IL USA), and anti-PARP and cleaved PARP (Cell Signaling).

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

Journal: Oncotarget

Article Title: Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer

doi: 10.18632/oncotarget.17527

Figure Lengend Snippet: ( A ) and ( B ) Colony formation assay in AGS. Panel (B) shows quantitative data of (A) (*** p < 0.001). ( C ) and ( D ) Colony formation assay in MKN45. Panel (D) shows quantitative data of (C) (* p < 0.05). ( E ) Organotypic 3D cell culture using MKN45 cells. Cells were harvested at day 4, 7 and 14. Results show HE staining of cells and display 3D (3-dimentional) structure. Tumor cells with GPX7 expression (Ad-GPX7) grew on the 3D culture at the beginning, but became much thinner at 1 week and finally died after 2 weeks, while control cells (Ad-ctrl) grew to form multiple layers at 2 weeks.

Article Snippet: The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, Proteintech Group, Chicago, IL USA), and anti-PARP and cleaved PARP (Cell Signaling).

Techniques: Colony Assay, Cell Culture, Staining, Expressing, Control

Journal: Oncotarget

Article Title: Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer

doi: 10.18632/oncotarget.17527

Figure Lengend Snippet: ( A ) EdU proliferation assay using AGS cells. Data shows GPX7-expressing cells (Ad-GPX7) had significantly lower EdU positive rate, meaning lower proliferation rate as compared to control cells (Ad-Ctrl). Right panel ( B ) shows quantitative data (*** p < 0.001). ( C ) Immunocytochemistry staining of p27 protein using fluorescence. Data shows that GPX7-expressing cells (Ad-GPX7) had significantly higher p27 expression than control cells (Ad-Ctrl). Right panel ( D ) displays quantitative data (* p < 0.05). ( E ) Immunocytochemistry staining of Cyclin D1 protein using fluorescence. Data shows that GPX7-expressing cells (Ad-GPX7) had significantly lower Cyclin D1 expression as compared to control cells (Ad-Ctrl). Right panel ( F ) displays quantitative data (*** p < 0.001).

Article Snippet: The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, Proteintech Group, Chicago, IL USA), and anti-PARP and cleaved PARP (Cell Signaling).

Techniques: Proliferation Assay, Expressing, Control, Immunocytochemistry, Staining, Fluorescence

Journal: Oncotarget

Article Title: Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer

doi: 10.18632/oncotarget.17527

Figure Lengend Snippet: ( A ) Immunocytochemistry staining of Ki67 protein using fluorescence. Data shows that GPX7-expressing cells (Ad-GPX7) had significantly lower Ki67 expression as compared to control cells (Ad-Ctrl). Right panel ( B ) displays quantitative data (*** p < 0.001). ( C ) Immunocytochemistry staining of p27 protein using fluorescence. Data shows that GPX7-expressing cells (Ad-GPX7) had significantly higher p27 expression as compared to control cells (Ad-Ctrl). Right panel ( D ) displays quantitative data (** p < 0.01). ( E ) Immunocytochemistry staining of Cyclin D1 protein using fluorescence. Data shows that GPX7-expressing cells (Ad-GPX7) had significantly lower Cyclin D1 expression as compared to control cells (Ad-Ctrl). Right panel ( F ) displays quantitative data (* p < 0.05). ( G ) and ( H ) show the HE images of organotypic culture corresponding to the fluorescence images.

Article Snippet: The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, Proteintech Group, Chicago, IL USA), and anti-PARP and cleaved PARP (Cell Signaling).

Techniques: Immunocytochemistry, Staining, Fluorescence, Expressing, Control

Journal: Oncotarget

Article Title: Glutathione peroxidase 7 suppresses cancer cell growth and is hypermethylated in gastric cancer

doi: 10.18632/oncotarget.17527

Figure Lengend Snippet: ( A ) Live cell images under an inverse microscope after reconstitution of GPX7 in AGS cells. Results show that tumor cells with GPX7 expression (Ad-GPX7) started to detach from the plate's bottom from day 3 of reconstitution of GPX7 and at day 6, most of the tumor cells were floating. ( B ) Trypan blue assay to count dead cells as well as viable cells. Data confirmed that the floating cells observed under microscope were dead cells. ( C ) Annexin V flow cytometry analysis of apoptosis. Data shows tumor cells with GPX7 expression (Ad-GPX7) displayed higher Annexin V cells ( p < 0.05). ( D ) Western blot analyses of cleaved PARP in AGS and MKN45 cells. Results confirmed that cells with GPX7 expression had more cleaved PARP in both cell lines. The western blots with molecular marker is shown in .

Article Snippet: The primary antibodies were: anti-GPX7 antibody (rabbit, 1:1000, Proteintech Group, Chicago, IL USA), and anti-PARP and cleaved PARP (Cell Signaling).

Techniques: Microscopy, Expressing, Flow Cytometry, Western Blot, Marker