783 research outputs found
Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI
No full textCell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create “PIP-FUCCI”. The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions.</p
Accumulation of Fucci proteins in tsTM3 cells at 39°C.
No full text<p>(A) Western blot analysis of Fucci in CHO-K1 or tsTM3 cells. Wild-type CHO-K1 or ts mutant tsTM3 cells expressing Fucci-G<sub>1</sub> Orange or Fucci-S/G<sub>2</sub>/M Green were grown at 34°C or incubated at 39°C for the times shown in the figure and lysed. The proteins resolved on acrylamide gels and were detected by immunoblotting with antibodies directed against fluorescent tags (mKO2 or mAG1) and Uba1. Only relevant parts of the blot are shown. α-tubulin is included as a loading control. Temperature-dependent increases in the amount of Fucci with the reduction of Uba1 were found in tsTM3 cells. (B) Quantitative analysis of bands in blots of tsTM3 cells expressing Fucci. Band intensities of Fucci and Uba1, like those shown in panel (A), were measured and are expressed relative to that of each at 34°C or relative to the lower band at 34°C with standard deviation of at least three experiments. <i>P</i> values were calculated by Student <i>t</i>-test. Significant differences from values at 34°C are shown by asterisks. Incubation at 39°C resulted in a significant increase of Fucci (mKO2-hCdt1 or mAG1-hGem) with decrease in Uba1A in tsTM3 cells.</p
Effects of incubation at 39°C on the activity of E1 analyzed with Fucci.
No full text<p>(A) Images of living cells expressing Fucci-G<sub>1</sub> Orange or Fucci-S/G<sub>2</sub>/M Green in wild-type CHO-K1 or ts mutant tsTM3 cells. Stable transformants expressing Fucci were grown at 34°C or incubated at 39°C for the times shown in the figure and analyzed. Small merged images with counterstain of Hoechst 33342 are embedded. Bar, 10 µm. (B) Quantitative analyses of the numbers of cells expressing Fucci in CHO-K1 and tsTM3 cells. Cells expressing Fucci, such as those shown in panel (A), were counted and are expressed as a ratio with standard deviation of at least three experiments. <i>P</i> values were calculated by Student <i>t</i>-test. Significant differences from values at 34°C are shown by asterisks. Incubation at 39°C increased the number of tsTM3 cells expressing Fucci signals.</p
The study of moral careers of ill person and patient for the implementation of partecipated care paths
No full textThe role of peroxisome proliferator-activated receptors in the esophageal, gastric, and colorectal cancer
No full textTumors of the gastrointestinal tract are among the most frequent human malignancies and account for approximately 30 of cancer-related deaths worldwide. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that control diverse cellular functions such as proliferation, differentiation, and cell death. Owing to their involvement in so many processes, they play crucial roles also in the development and physiology of the gastrointestinal tract. Consistently, PPARs deregulation has been implicated in several pathophysiological conditions, including chronic inflammation and cancer development. This paper summarizes the current knowledge on the role that the various PPAR isoforms play in the pathogenesis of the esophageal, gastric, and intestinal cancer. Elucidation of the molecular mechanisms underlying PPARs' signaling pathways will provide insights into their possible use as predictive biomarkers in the initial stages of the process. In addition, this understanding will provide the basis for new molecular targets in cancer therapy and chemoprevention. Copyright © 2012 Alessandra Fucci et al
Characterization of Fucci-expressing NMuMG cells.
No full text<p>(<b>a–f</b>) Flow cytometry analysis of Ki-67- and DAPI-stained NMuMG/Fucci cells. Fucci signals (mKO2 vs. mAG) of exponentially growing cells (<b>a</b>) and cells at confluency (<b>d</b>). Cells collected in gate #1 were found to be diploid (2 C) and Ki-67 negative, and thus were assigned to quiescent G<sub>0</sub> phase. (<b>g</b>) Fluorescence images of NMuMG/Fucci cells, after a “wound” (scratch) was introduced into a confluent monolayer. Scale bar, 10 µm. (<b>h, i</b>) Time-lapse imaging (<b>h</b>) and fluorescence intensity (<b>i</b>) of mKO2-hCdt1(30/120) (red line) and mAG-hGem(1/110) (green line) in the cell indicated by the white arrowhead in (<b>g</b>).</p
Characterization of abnormal Fucci fluorescence following KPU-300 treatment.
No full text<p>(A) Representative images of abnormal fluorescence after treatment with KPU-300. The time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. Bar, 20 μm (B) Relationship between abnormal Fucci fluorescence and M phase following KPU-300 treatment. (a) Two-dimensional flow-cytometric analysis of Fucci fluorescence. The area within a quadrangle represents cells expressing abnormal Fucci fluorescence. (b) Two-dimensional flow-cytometric analysis of DNA content and phosphorylated histone H3 (pHH3). The area within a quadrangle represents cells in M phase. The acquired time points are shown as hours:minutes in each image; 0:00 represents the start of drug treatment. (c) Quantitative analysis of cells with abnormal Fucci expression and those in M phase in Fig 3a and 3b. Data represent means ± S.E. of values obtained from three independent experiments. *<i>p</i> < 0.05; **<i>p</i> < 0.01 vs. controls at time 0.</p
Fly-FUCCI: A versatile tool for studying cell proliferation in complex tissues
No full textOne promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent ubiquitination-based cell cycle indicator). Here, we report the development of a Drosophila-specific FUCCI system (Fly-FUCCI) that allows one to distinguish G1, S, and G2 phases of interphase. Fly-FUCCI relies on fluorochrome-tagged degrons from the Cyclin B and E2F1 proteins, which are degraded by the ubiquitin E3-ligases APC/C and CRL4Cdt2, during mitosis or the onset of S phase, respectively. These probes can track cell-cycle patterns in cultured Drosophila cells, eye and wing imaginal discs, salivary glands, the adult midgut, and probably other tissues. To support a broad range of experimental applications, we have generated a toolkit of transgenic Drosophila lines that express the Fly-FUCCI probes under control of the UASt, UASp, QUAS, and ubiquitin promoters. The Fly-FUCCI system should be a valuable tool for visualizing cell-cycle activity during development, tissue homeostasis, and neoplastic growth
Results of the Fifth International Spectroradiometer Comparison for Improved Solar Spectral Irradiance Measurements and Related Impact on Reference Solar Cell Calibration
No full textThis paper reports on the results of the fifth spectral irradiance measurement intercomparison and the impact these results have on the spread of spectral mismatch calculations in the outdoor characterization of reference solar cell and photovoltaic (PV) devices. Ten laboratories and commercial partners with their own instruments were involved in the comparison. Solar spectral irradiance in clear sky condition was measured with both fast fixed and slow rotating grating spectroradiometers. This paper describes the intercomparison campaign, describes different statistical analysis used on acquired data, reports on the results, and analyzes the impact these results would have on the primary calibration of a c-Si PV reference cell under natural sunlight
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