cell cycle indicator fucci  (TaKaRa)

Journal: International Journal of Oncology

Article Title: Discovery of a potent cytotoxic agent that promotes G 2 /M phase cell cycle arrest and apoptosis in a malignant human pharyngeal squamous carcinoma cell line

doi: 10.3892/ijo.2022.5331

Figure Lengend Snippet: Detroit 562 cells arrested in the G2 phase are susceptible to cell death. (A) Examination of the cell cycle in response to 24a treatment. Cell cycle distribution was determined by flow cytometric analysis of PI-stained cells following 24-h treatment at the indicated doses. GM04390 is normal human fibroblasts. Values represent the mean of n=2. (B) Examination of real-time cell cycle progression using FastFUCCI-expressing Detroit 562 cells. A fusion protein of Cdt1 and monomeric Kusabira Orange 2 creates the FUCCI reporter that allows visualization of cells in the G1 phase. A fusion of geminin with monomeric Azami Green allows for the monitoring of cells in the G2 phase. Cells were treated with 0.03 µM 24a for the indicated duration and PI was added 1 h prior to imaging. As shown circled, only cells arrested in the G2 phase also stained for PI. Magnification, ×20. (C) Assessment of compound binding by isothermal titration calorimetry. Random, fragmented DNA was titrated into a sample of buffer, 60 µM EtBr or 100 µM 24a. Raw heat values without further deconvolution are demonstrated. PI, propidium iodide; EtBr, ethidium bromide; FUCCI, fluorescence ubiquitination-based cell cycle indicator.

Article Snippet: Construction of Detroit 562 cells expressing fluorescence ubiquitination-based cell cycle indicator (FUCCI) VSV-G pseudotyped lentiviral particles were generated from the transfection of 293T cells (ATCC) by the Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's recommendation with the 3rd generation pBOB-EF1-FastFUCCI-Puro transfer vector (plasmid no. 86849), the 2nd generation packaging plasmid pCMV-dR8.2 dpr (plasmid no. 8455) and the 2nd generation envelope plasmid pCMV-VSV-G (plasmid no. 8454) obtained from Addgene, Inc. 293T cells (at ~95% confluence in a 10-cm dish) were transfected with 10 μ g transfer vector, 5 μ g packaging plasmid and 5 μ g envelope plasmid and the media harvested 48 and 72 h post-transfection.

Techniques: Staining, Expressing, Imaging, Binding Assay, Isothermal Titration Calorimetry, Fluorescence, Ubiquitin Proteomics

Journal: Journal of Dental Sciences

Article Title: Rho GTPase activating protein 11A promotes tongue squamous cell carcinoma proliferation and is a transcriptional target of forkhead box M1

doi: 10.1016/j.jds.2024.02.015

Figure Lengend Snippet: ARHGAP11A knockdown induces G1 phase arrest by regulating multiple cell-cycle proteins in tongue squamous cell carcinoma. A-B. Representative fluorescent ubiquitination-based cell cycle indicator (FUCCI) images and ARHGAP11A staining (A) and quantitation of ARHGAP11A mRNA and protein expression in different phases with the cell-cycle (B) in U2OS cells in the human protein atlas (HPA). Image credit: the HPA, data were retrieved from: https://www.proteinatlas.org/ENSG00000198826-ARHGAP11A/subcellular#cell_cycle . C. Western blotting analysis was performed to check the expression of indicated proteins in SCC4 and SCC25 cells 48 h after lentivirus-mediated ARHGAP11A knockdown. D-E. Representative images (D) and quantitation (E) of cell-cycle distribution of SCC4 and SCC25 cells with or without lentivirus-mediated ARHGAP11A knockdown. ∗∗∗, P < 0.001.

Article Snippet: Our review of fluorescent ubiquitination-based cell cycle indicator (FUCCI) staining data from the Human Protein Atlas (HPA) revealed that cells in the G2/M phase (Geminin-positive) exhibited stronger ARHGAP11A staining compared to those in the G1 phase (CDT1-positive) ( A).

Techniques: Knockdown, Staining, Quantitation Assay, Expressing, Western Blot

Journal: Journal of Dental Sciences

Article Title: Rho GTPase activating protein 11A promotes tongue squamous cell carcinoma proliferation and is a transcriptional target of forkhead box M1

doi: 10.1016/j.jds.2024.02.015

Figure Lengend Snippet: ARHGAP11A knockdown induces G1 phase arrest by regulating multiple cell-cycle proteins in tongue squamous cell carcinoma. A-B. Representative fluorescent ubiquitination-based cell cycle indicator (FUCCI) images and ARHGAP11A staining (A) and quantitation of ARHGAP11A mRNA and protein expression in different phases with the cell-cycle (B) in U2OS cells in the human protein atlas (HPA). Image credit: the HPA, data were retrieved from: https://www.proteinatlas.org/ENSG00000198826-ARHGAP11A/subcellular#cell_cycle . C. Western blotting analysis was performed to check the expression of indicated proteins in SCC4 and SCC25 cells 48 h after lentivirus-mediated ARHGAP11A knockdown. D-E. Representative images (D) and quantitation (E) of cell-cycle distribution of SCC4 and SCC25 cells with or without lentivirus-mediated ARHGAP11A knockdown. ∗∗∗, P < 0.001.

Article Snippet: Representative fluorescent ubiquitination-based cell cycle indicator (FUCCI) images and ARHGAP11A staining (A) and quantitation of ARHGAP11A mRNA and protein expression in different phases with the cell-cycle (B) in U2OS cells in the human protein atlas (HPA).

Techniques: Knockdown, Ubiquitin Proteomics, Staining, Quantitation Assay, Expressing, Western Blot