nucleotide guide rnas  (Axolabs Inc)

Journal: Molecular Biology of the Cell

Article Title: Epithelial tissue geometry directs emergence of bioelectric field and pattern of proliferation

doi: 10.1091/mbc.e19-12-0719

Figure Lengend Snippet: Figure 3: Gradients of Vm and proliferation require intercellular contacts. (A) Immunoblotting analysis for E-cadherin (Ecad) and GAPDH in parental mammary epithelial and Enull cells (n=3). (B) Enull cells were microfabricated into square tissues and stained for nuclei and β-catenin. (C) EdU analysis of an individual Enull tissue and (D) frequency maps of 25 tissues across a representative replicate reveal a uniform pattern of DNA synthesis. (E) DiBac4(3) staining of an individual Enull tissue and (F) frequency map of 27 tissues across a representative replicate. (G) Quantification of DiBac4(3) fluorescence in different regions of Enull tissues (n=3 independent replicates). Shown are mean + SD. *, P<0.05; **, P<0.01; ***, P<0.001, as determined by an unpaired parametric t-test with Welch’s correction. (H) Traction force microscopy of Enull tissues (n=3) revealed no discernable pattern of mechanical stress within the tissue. Scale bars represent 50 m.

Article Snippet: Specifically, cells were transfected either with plasmid encoding the Cas9 nuclease and a 20-nucleotide guide RNA targeting CDH1 (sc-419587, Santa Cruz, Dallas, TX) or control CRISPR/Cas9 plasmid (sc-418922, Santa Cruz) co-expressing GFP.

Techniques: Western Blot, Staining, DNA Synthesis, Fluorescence, Microscopy

Journal: Nature Chemical Biology

Article Title: Paradoxical mitotic exit induced by a small molecule inhibitor of APC/CCdc20

doi: 10.1038/s41589-020-0495-z

Figure Lengend Snippet: Fig. 5 | p31comet and apcin cooperate to reactivate APC/C. a, HCT116 cells were treated with 5 nM control or p31comet-targeting siRNA, followed by combinations of nocodazole, apcin (0, 6.2, 12.5, 25 or 50 μM) and AZ3146 (0, 0.25, 0.5, 1 or 2 μM) for 24 h. The fraction of mitotic cells was determined with a high-throughput fixed cell assay. The mean of four biological replicates from two independent experiments is shown; error bars, s.d. Statistical significance was calculated by two-tailed unpaired t test (*P < 0.05, ***P < 0.001, ****P < 0.0001). Exact P values are shown in Supplementary Table 2. b, Reactions were carried out in the presence of APC/C, 3×Myc-His–Cdc20-A, Ube2S, Ube2C, E1, WT ubiquitin (Ub) and ATP ± MCC containing 3×FLAG– Cdc20-M ± apcin (500 μM) ± p31comet (1 μM). All components (except substrate) were preincubated for 5 min followed by addition of fluorescently labeled cyclin A (CycA*). Ubiquitination was analyzed by fluorescence imaging. The experiment was repeated independently with similar results. c, APC/C carrying a Strep tag on APC4 was incubated with Strep-Tactin resin, 3×Myc-His–Cdc20-A and MCC containing 3×Flag–Cdc20-M ± p31comet (1 μM) ± apcin or apcin-M (500 μM) for 1 h, and eluates were analyzed by immunoblotting. d, Quantification of the experiment in c. Protein abundance was normalized to that of Cdc27. The fraction of bound protein was set to 1 for the condition without compound. The mean of five independent experiments is shown; error bars, s.e.m. Statistical significance was calculated by two-tailed unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). e, MCC containing 3×FLAG–Cdc20-M was incubated with anti-FLAG beads and 3×Myc-His–Cdc20-A ± p31comet (1 μM) ± apcin or apcin-M (500 μM) for 1 h, and eluates were analyzed by immunoblotting. f, Quantification of the experiment in e. The mean of five independent experiments is shown. Quantification and representation are identical to d, except that protein abundance was normalized to that of 3×FLAG–Cdc20-M. Uncropped images for b,c,e are shown in Supplementary Fig. 8. Exact P values for d,f are shown in Supplementary Table 2.

Article Snippet: A 20-nucleotide guide RNA (gRNA) for CDC20 (5′-GGCGCATCCAGGATACGGTC-3′) was cloned into pSpCas9(BB)2A-Puro (PX459) v2.0 CRISPR–Cas9 vector (Addgene 62988) and confirmed by Sanger sequencing.

Techniques: Control, High Throughput Screening Assay, Two Tailed Test, Ubiquitin Proteomics, Labeling, Fluorescence, Imaging, Strep-tag, Incubation, Western Blot, Quantitative Proteomics