Journal: eLife

Article Title: Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor

doi: 10.7554/eLife.45116

Figure Lengend Snippet:

Article Snippet: Transfected construct ( H. sapiens ) , SNAP-B2AR , modified from addgene plasmid pSNAPf-ADRβ2 , Addgene Plasmid #101123 , .

Techniques: Transfection, Construct, Modification, Plasmid Preparation, Mutagenesis, Synthesized, Software

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