Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer

doi: 10.1186/s13046-023-02689-y

Figure Lengend Snippet: Engineered p16 epimutation recapitulates key features of age-associated epigenetic silencing. A p16 DNA methylation profiles in cis -MEF following serial passaging (p). A schematic of the p16 promoter of the cis -element knock-in allele with the CpG maps is shown. B Kinetics of p16 promoter methylation and mRNA expression in ctr-MEF and cis -MEF. Methylation levels were averaged from CpGs from − 814 bp to − 589 bp relative to the TSS. Note that cis -MEF cells grew for more than 25 passages; in contrast, the controls (ctr-MEF) entered growth arrest at passage 8 (p8) via p16 up-regulation. The p16 gene expression is relative to β–actin. C UCSC Genome Browser tracks showing the DNA methylation status of p16 , p19 , and p15 in the INK4/ARF locus on chromosome 4. The chromosomal coordinates are annotated on the top. The WGBS tracks show DNA methylation profiling in ctr-MEF (p7) and cis-MEF (p10). The height of each bar represents the methylation level of an individual CpG between 0 and 1 (100%). Promoters are indicated by angled arrows, and the knock-in location is indicated by an asterisk. D Comparisons of p16 promoter methylation in cis -MEF before and after Ad-Cre mediated cis -element excision with increasing doses of DAC. The culture passage number of cis -MEF is at p23. E Side-by-side comparisons of p16 expression in cis -MEF under the same conditions as ( D ). For ( D) and ( E ), data are shown as mean ± SEM with individual values from three independent experiments. P values were determined by a one-way ANOVA test. F dCas9-SunTagTET1-mediated targeted demethylation of p16 in cis -MEF. p16 -gRNA indicates GFP-positive cells treated with a p16 -specific gRNA that binds the promoter region from − 792 bp to − 769 bp relative to the TSS. Ctr indicates GFP-negative cells without targeted demethylation. Data are shown from three independent experiments. G Targeted promoter demethylation resulted in p16 gene reactivation. The culture passage number of cis -MEF is at p23. P values were determined by a two-tailed Student’s t -test

Article Snippet: Plasmids containing p16 -specific gRNA or non-targeting control gRNA were constructed using an all-in-one vector from Addgene (#82,559; Watertown, MA, USA) and Gibson Assembly (NEB, Ipswich, MA, USA). gRNA sequences are listed in Supplementary Table .

Techniques: DNA Methylation Assay, Passaging, Knock-In, Methylation, Expressing, Gene Expression, Two Tailed Test

Journal: International journal of molecular sciences

Article Title: Epigenetic Regulation of MAP3K8 in EBV-Associated Gastric Carcinoma.

doi: 10.3390/ijms24031964

Figure Lengend Snippet: Figure 2. Super-enhancers regulate MAP3K8 expression in EBVaGC. (A,B) Four CRISPRi plas- mids corresponding to four MAP3K8 SE regions located between (a) 30,706,373–30,709,258 kb (Region 1); (b) 30,711,625–30,713,797 kb (Region 2); (c) 30,716,284–30,717,068 kb (Region 3); and (d) 30,816,494–30,819,552 kb (Region 4) of chromosome 10 were formulated. EBVaGC cells were transfected with these plasmids to create MAP3K8 SE repressed EBVaGC stable cell lines. RNA was isolated 48 h after selection. The relative mRNA levels of MAP3K8 in MAP3K8 SE repressed SNU719 (A) and YCCEL1 (B) cells using qRT-PCR. MAP3K8 expression levels were normalized to the housekeeping gene GAPDH. (C,D) Proliferation of MAP3K8 SE repressed SNU719 (C) and YCCEL1 (D) cells were determined by MTT. An MTT was performed at 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. (E,F) mRNA (E) and protein (F) levels of MAP3K8 in OTX015 treated SNU719 cells. SNU719 cells were treated with 20 µM OTX015 and RNA and the total proteins were isolated at the same time (48 h post-treatment) for PCR and WB. (G) Proliferation of OTX015 treated SNU719 cells. SNU719 cells were treated with 20 µM OTX015 and an MTT was performed at 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. (H,I) mRNA (H) and protein (I) levels of MAP3K8 in OTX015 treated YCCEL1 cells. YCCEL1 cells were treated with 20 µM OTX015 and RNA and total proteins were isolated simultaneously (48 h post-treatment) for PCR and WB. (J) Proliferation of OTX015 treated YCCEL1 cells. YCCEL1 cells were treated with 20 µM OTX015 and an MTT was performed at 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. Three and four biological repeats were performed for the qRT-PCR and MTT, respectively. All experiments were repeated a minimum of three times and the data are represented as the mean ± SD (ns = non-significant, *** p ≤0.001, ** p ≤0.01, and * p ≤0.05).

Article Snippet: HEK293T cells were then transfected with CRISPRi plasmid, lentiviral packaging plasmid (psPAX2, Addgene, catalogue no.: 12260), and VSV-G envelope expressing plasmid (pMD2.G, Addgene, catalogue no.: 12259), simultaneously, using a lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, catalogue no.: L3000-008).

Techniques: Expressing, Transfection, Stable Transfection, Isolation, Selection, Quantitative RT-PCR