Journal: PLoS ONE
Article Title: The Human Homolog of Escherichia coli Endonuclease V Is a Nucleolar Protein with Affinity for Branched DNA Structures
Figure Lengend Snippet: Upregulation of ENDOV transcription during quiescence. ( A ) Human embryonic fibroblasts were arrested in G0 by serum starvation at confluence and released by replating 1∶4 in culture medium with serum. ENDOV transcript levels (exons 2 to 3) were measured during cell cycle progression after G0 release at indicated time points by qRT-PCR. G0 cells were used as the reference for calculations. The average of 3 parallels (same RNA) was calculated and standard deviation is shown. Cell cycle distribution was monitored using propidium iodide staining followed by flow cytometry after release from the block. The percentage of cells in each cell cycle is presented in the table. The experiment was repeated twice with similar results. ( B ) Nothern blot analysis of ENDOV mRNA. mRNA was isolated from human fibroblasts that were unsynchronised (u), G0 arrested (G0) and allowed to proliferate for 24 hours (t 24 ), separated by electrophoresis and transferred to a nylon membrane. Hybridisation signals with probes spanning exons 4–8, exon 10, exon 3 of ENDOV and for β -ACTIN are shown. M is the RNA size standard as indicated (in kilobases).
Article Snippet: Cell cycle synchronization and analysis by flow cytometrySynchronization of the cells in G0 phase was achieved by culturing cells as a confluent layer for 72 h followed by serum starvation (0.2% serum) for 72 h. The cells were released from G0 by trypsination (Trypsin-EDTA 200 mg/l, Lonza) for 4 min at 37°C and cultivated in standard growth medium at 25% confluence.
Techniques: Quantitative RT-PCR, Standard Deviation, Staining, Flow Cytometry, Blocking Assay, Isolation, Electrophoresis, Hybridization