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: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/eLife.66095

Figure Lengend Snippet: ( A ) Cas9-expressing H322 cells (H322-Cas9) were transduced with lentiviruses containing non-targeting gRNA (gNT) or two gRNAs targeting LKB1 (gLKB1-1, gLKB1-2). H322 gNT cell lysates were treated with or without alkaline calf intestinal phosphatase (CIP, one unit per ug protein) for 60 min at 37°C. The samples were loaded together with untreated cell lysates in 6% gels for immunoblotting for CRTC1, CRTC2, CRTC3, and LKB1. Blotting with anti-β-Tubulin was used as a loading control. ( B ) Nuclear and cytoplasmic fractions of H322-Cas9 containing gNT, gLKB1-1, or gLKB1-2 cells were isolated for immunoblotting to detect three CRTCs and LKB1. HDAC1 and β-Tubulin were detected as nuclear or cytoplasmic controls, respectively. Figure 1—figure supplement 3—source data 1. Unedited immunoblots in A, B.

Article Snippet: Recombinant DNA reagent , non-targeting control gRNA (plasmid) , Addgene , Plasmid #80180 , .

Techniques: Expressing, Transduction, Western Blot, Control, Isolation

Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/eLife.66095

Figure Lengend Snippet: The Cas9-expressing H322 cells (H322-Cas9) were first generated and then transduced with lentiviruses containing non-targeting gRNA (gNT) or two gRNAs targeting LKB1 (gLKB1-1, gLKB1-2). The transcript levels of several top dnCRTC-regulated genes (INSL4, CPS1, NR4A2, and PDK4) in these cells were determined by RT-qPCR. The LKB1-null NSCLC A549 cells were also analyzed. It is noted that the upregulation of these genes was moderate as compared to their corresponding levels in LKB1-null NSCLC A549 cells. Assays were performed in triplicate. The p values show one-way ANOVA tests between LKB1 knockout samples and control samples. *p<0.05, ***p<0.001, ns p>0.05. Figure 4—figure supplement 2—source data 1. Numerical data for bar graphs.

Article Snippet: Recombinant DNA reagent , non-targeting control gRNA (plasmid) , Addgene , Plasmid #80180 , .

Techniques: Expressing, Generated, Transduction, Quantitative RT-PCR, Knock-Out, Control

Journal: eLife

Article Title: Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

doi: 10.7554/eLife.66095

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , non-targeting control gRNA (plasmid) , Addgene , Plasmid #80180 , .

Techniques: Recombinant, Plasmid Preparation, Sequencing, Clone Assay, Control, Cell Culture, Transfection, RNA Extraction, Reverse Transcription, SYBR Green Assay, Extraction, Reporter Assay, Software