the sequenom epityper massarray system  (Sequenom)

Journal: Therapeutic Advances in Medical Oncology

Article Title: The role of aberrant DNA methylation in cancer initiation and clinical impacts

doi: 10.1177/17588359231220511

Figure Lengend Snippet: Summary of methods available for tumour suppressor methylation testing.

Article Snippet: Sequenom MassARRAY (EpiTYPER) , Bisulphite DNA , PCR of bisulphite DNA followed by base-specific cleavage and MALDI-TOF , High-throughput, (semi-) quantitative analysis of multiple CpG sites , Only average methylation level per CpG position.

Techniques: Methylation, Nucleic Acid Electrophoresis, DNA Methylation Assay, Amplification, Electrophoresis, Denaturing Gradient Gel Electrophoresis, High Performance Liquid Chromatography, Binding Assay, Radioactivity, Fluorescence, Combined Bisulfite Restriction Analysis Assay, Sequencing, Multiplexing, Microarray, Genome Wide, Bisulfite Sequencing, Nanopore Sequencing, Sampling, Methylated DNA Immunoprecipitation, Immunoprecipitation

Journal: Acta Pharmaceutica Sinica. B

Article Title: UHRF1/DNMT1–MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

doi: 10.1016/j.apsb.2023.02.015

Figure Lengend Snippet: Differential CpG methylation in association with PRSS3 transcript expression in lung cancer cells. (A) Schematic diagram of the PRSS3 gene structure. (B, C) The correlation between methylation and PRSS3 transcript expression was visualized using a distance function for heatmap cluster analysis of methylation in lung cancer cell lines (B) or in extended fragments in lung cancer tissue specimens ( n = 315) (C). In the heatmap, the columns indicate cell lines or tissue specimens. Before correlation analysis, both the transcript and DNA methylation levels of PRSS3 were renormalized with a mean = 0 and a standard deviation = 1. The statistical significance of correlation coefficients between mRNA expression of the PRSS3 transcripts and a CpGI (B) or CpG site (C) are shown at the bottom (see Fig. S9 for details). ∗ P < 0.05, ∗∗ P < 0.01. (D) MSP-qPCR analysis of PRSS3 iCpGI methylation in NSCLC cells. In vitro methylated DNA (IVD) and normal blood lymphocyte DNA (NL) served as positive and negative controls, respectively. (E) MSP-qPCR of PRSS3 iCpGI methylation frequency in tumor and adjacent tissue samples from NSCLC patients ( n = 13). The frequencies of methylation (M) and unmethylation (UM) in the samples are presented as percentages of cases. ∗∗ P < 0.01 (unpaired t test). (F, G) Quantitative analysis of CpG site methylation along the PRSS3 DMR using MassARRAY assays. The differential CpG site methylation status in PRSS3 is shown as the average methylation level (F) and a stacked line (top lane) linked to a heatmap graph (bottom lane) (G) in lung cancer cells. The regions covered by the assays are described as MassARRAY_R1, _R2 and _R3. Methylated and nonmethylated DNA from human HCT116 cells with DNMT1 and DNMT3B knockout were used as methylated and unmethylated control template DNA, respectively. An R package was used to create the cluster heatmap visualization. The constitutively methylated CpGs in the intragenic CpG island (iCpGI) are shown. (H, I) RT-qPCR analysis of the expression of PRSS3 (H) and PRSS3-V1 – V3 (I) in NSCLC cell lines treated with 5-Aza-CdR (0.5 μmol/L, 96 h). The results represent the mean ± SD (standard deviation). Differences in PRSS3 expression were analyzed using a t test (H) and are presented as the difference in mean proportion (%) for PRSS3-V1 – V3 using the R package (I). The data are representative of at least three independent experiments. The data are shown as the mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01. (J) MassARRAY monitoring the alteration in PRSS3 iCpGI methylation in NSCLC cells upon 5-Aza-CdR treatment (5 μmol/L, 96 h).

Article Snippet: MassARRAY EpiTYPER (Sequenom) was used for data analysis to generate quantitative results for each CpG site or an aggregate of the results for multiple CpG sites.

Techniques: CpG Methylation Assay, Expressing, Methylation, DNA Methylation Assay, Standard Deviation, In Vitro, Knock-Out, Control, Quantitative RT-PCR

Journal: Acta Pharmaceutica Sinica. B

Article Title: UHRF1/DNMT1–MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

doi: 10.1016/j.apsb.2023.02.015

Figure Lengend Snippet: Regulation of PRSS3 transcripts by the UHRF1/DNMT1 complex. (A) The expression of DNMTs (DNMT1, DNMT3A and DNMT3B) in NSCLC cells was analyzed using RT-qPCR and Western blotting. (B) Schematic representation of part of PRSS3 gene with marked positions of primers for MeDIP-qPCR/ChIP-qPCR and MassARRY assay. (C) MeDIP-qPCR analysis of anti-5-mC-enriched DNA fragments associated with PRSS3 in A549 cells stably transfected with DNMT1 (left panel) and in NCI-H460 cells with DNMT1 knockdown or 5-Aza-CdR treatment (0.5 μmol/L, 96 h) (right panel). Specific enrichment is presented as the percentage of the input (% of input). (D) MassARRAY analysis of PRSS3 iCpGIm. (E) RT-qPCR and Western blot analysis of UHRF1 expression in NSCLC cells. (F) Western blot of DNMT1 levels following UHRF1 overexpression in A549 cells. (G) A ChIP assay with an anti-UHRF1 antibody was performed on UHRF1 -overexpressing A549 cells. Immunoprecipitated genomic DNA was analyzed using qPCR specific for PRSS3 iCpGI. Specific enrichment is presented as the percentage of the input (% of input). (H) ChIP-qPCR analysis of DNA fragments enriched by anti-DNMT1 or anti-UHRF1 antibody and associated with PRSS3 iCpGI in A549 cells with DNMT1 overexpression (left panel) and NCI-H460 cells with DNMT1 knockdown (right panel). (I) The mRNA levels of PRSS3 transcripts in stable DNMT1- overexpressing A549 cells and DNMT1 -knockdown H460 cells. (J, K) Western blot (J) and immunofluorescence (K) analyses of the expression of the PRSS3 isoforms following UHRF1 transfection in A549 cells. DAPI-stained nuclei are shown in blue. Scale bar: 1 μm. The data are representative of at least three independent experiments. The data are shown as the mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01 indicate significant differences from the control by t test.

Article Snippet: MassARRAY EpiTYPER (Sequenom) was used for data analysis to generate quantitative results for each CpG site or an aggregate of the results for multiple CpG sites.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Methylated DNA Immunoprecipitation, ChIP-qPCR, Stable Transfection, Transfection, Knockdown, Over Expression, Immunoprecipitation, Immunofluorescence, Staining, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: UHRF1/DNMT1–MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

doi: 10.1016/j.apsb.2023.02.015

Figure Lengend Snippet: DATS interacted with 5-Aza-CdR to regulate PRSS3 expression via site-specific iCpGI methylation. (A) A549 cells were treated with different concentrations of diallyl trisulfide (DATS, 12.5, 25 and 50 μmol/L). Cell proliferation and migration were measured using MTT (left panel), colony formation (middle panel) and Transwell (right panel) assays, with quantification data presented in the top panels and representative images in the bottom panels. (B) The expression of PRSS3-SVs in NSCLC cells treated with different concentrations of DATS was analyzed by RT-qPCR (left panel) and Western blotting (right panel). (C) RT-qPCR results (left panel) and Western blot (right panel) showing the expression of DNMTs (DNMT1, DNMT3A and DNMT3B) in NSCLC cells treated with different concentrations of DATS. (D) PRSS3 iCpGI association with UHRF1/DNMT1 in A549 cells upon 50 μmol/L DATS treatment was measured by ChIP-qPCR using anti-DNMT1 or anti-UHRF1 antibody. (E) The enrichment of methylated PRSS3 iCpGI fragments in A549 cells after 50 μmol/L DATS treatment was analyzed by MeDIP-qPCR using an anti-5mC antibody. (F) A549 cells were treated with 50 μmol/L DATS for 12 h and then transfected with PRSS3-SVs ( PRSS3-V1 – V3 ). MTT and Transwell assays were used to examine cell proliferation and migration (top: quantification data; bottom: representative images). (G–I) A549 cells were treated with DATS (50 μmol/L) combined with 5-Aza-CdR (0.5 μmol/L) or transfected with MZF1 L . MTT and Transwell assays were conducted to monitor cell proliferation and migration ( top : quantification data; bottom : representative images) (G). The expression of MZF1 L and PRSS3-V3 was analyzed by RT-qPCR (left panel) and Western blotting (right panel) (H). ChIP-qPCR analysis of PRSS3 iCpGI genomic DNA enriched by anti-MZF1 antibody (I). (J–K). Summary table showing CpG site-specific methylation status in PRSS3 iCpGI quantitatively measured with a MassARRAY assay. Red fronts: hypermethylation; black fronts: hypomethylation (J). Ranking analysis of the demethylation efficiency of DATS/5-Aza-CdR in lung cancer cells (K). (L) A549 cells ectopically expressing MZF1 L or vector control were transplanted subcutaneously into the left and right flanks of nude mice ( n = 2 flanks × 4 mice in each group). Eight days after implantation of A549 cells, the mice were treated with DATS (i.p., 20 mg/kg) and/or 5-Aza-CdR (i.p., 0.5 mg/kg) or saline once per week. Left panel : tumor volumes in tumor-bearing mice measured at the indicated time points. Middle panel : Images of A549 xenograft tumors dissected from nude mice on Day 32. Right panel : Body weights of the mice. P values were calculated via one-way ANOVA with Tukey's post hoc test. The data are representative of at least three independent experiments. The data are shown as the mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01 indicate significant differences from the control, NS, no significance.

Article Snippet: MassARRAY EpiTYPER (Sequenom) was used for data analysis to generate quantitative results for each CpG site or an aggregate of the results for multiple CpG sites.

Techniques: Expressing, Methylation, Migration, Quantitative RT-PCR, Western Blot, ChIP-qPCR, Methylated DNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, Saline