The distribution of fluorescence signals measured with flow cytometry can be influenced by several factors, including qualitative and quantitative properties of the used fluorochromes, optical properties of the detection system, as well as the variability within the analyzed cell population itself. Most of the single cell samples prepared from in vitrocultures or clinical specimens contain a variable cell cycle component. Cell cycle, together with changes in the cell size, are two of the factors that alter the functional properties of analyzed cells and thus affect the interpretation of obtained results. Here, we describe the association between cell cycle status and cell size, and the variability in the distribution of fluorescence intensity as determined with flow cytometry, at population scale. We show that variability in the distribution of background and specific fluorescence signals is related to the cell cycle state of the selected population, with the 10% low fluorescence signal fraction enriched mainly in cells in their G0/G1 cell cycle phase, and the 10% high fraction containing cells mostly in the G2/M phase. Therefore we advise using caution and additional experimental validation when comparing populations defined by fractions at both ends of fluorescence signal distribution to avoid biases caused by the effect of cell cycle and cell size.
- MeSH
- buněčné dělení MeSH
- buněčný cyklus fyziologie MeSH
- G2 fáze * MeSH
- průtoková cytometrie metody MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.
- MeSH
- buněčný cyklus fyziologie MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- fosfoproteiny chemie metabolismus MeSH
- fosforylace MeSH
- glutamáty metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- izotopové značení metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- peptidy metabolismus MeSH
- peroxiredoxin VI chemie metabolismus MeSH
- proteolýza * MeSH
- proteom genetika metabolismus MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- sestřihové faktory chemie metabolismus MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The cytoskeleton plays a key role in cellular proliferation, cell-shape maintenance and internal cellular organization. Cells are highly sensitive to changes in microgravity, which can induce alterations in the distribution of the cytoskeletal and cell proliferation. This study aimed to assess the effects of simulated microgravity (SMG) on the proliferation and expression of major cell cycle-related regulators and cytoskeletal proteins in human umbilical cord mesenchymal stem cells (hucMSCs). A WST-1 assay showed that the proliferation of SMG-exposed hucMSCs was lower than a control group. Furthermore, flow cytometry analysis demonstrated that the percentage of SMG-exposed hucMSCs in the G0/G1 phase was higher than the control group. A western blot analysis revealed there was a downregulation of cyclin A1 and A2 expression in SMG-exposed hucMSCs as well. The expression of cyclin-dependent kinase 4 (cdk4) and 6 (cdk6) were also observed to be reduced in the SMG-exposed hucMSCs. The total nuclear intensity of SMG-exposed hucMSCs was also lower than the control group. However, there were no differences in the nuclear area or nuclear-shape value of hucMSCs from the SMG and control groups. A western blot and quantitative RT-PCR analysis showed that SMG-exposed hucMSCs experienced a downregulation of bata-actin and alpha-tubulin compared to the control group. SMG generated the reorganization of microtubules and microfilaments in hucMSCs. Our study supports the idea that the downregulation of major cell cycle-related proteins and cytoskeletal proteins results in the remodeling of the cytoskeleton and the proliferation of hucMSCs.
- MeSH
- buněčná diferenciace fyziologie MeSH
- buněčný cyklus fyziologie MeSH
- cytoskelet metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie metabolismus MeSH
- mikrofilamenta metabolismus MeSH
- mikrotubuly metabolismus MeSH
- proliferace buněk fyziologie MeSH
- pupečník cytologie metabolismus MeSH
- simulace stavu beztíže * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Aging and tumorigenesis are associated with decline and disruption of circadian rhythms in many tissues and accumulating evidence indicates molecular link between circadian clock and cell cycle. The aim of this study was to investigate the effect of aging and tumorigenesis on coupling between cell cycle and circadian clock oscillators in colon, which undergoes regular rhythmicity of cell cycle and expresses peripheral circadian clock. Using healthy 14-week-old mice and 33-week-old mice with and without colorectal tumors, we showed that the 24-h expression profiles of clock genes and clock-controlled genes were mostly unaffected by aging, whereas the genes of cell cycle and cell proliferation were rhythmic in the young colons but were silenced during aging. On the other hand, tumorigenesis completely silenced or dampened the circadian rhythmicity of the clock genes but only a few genes associated with cell cycle progression and cell proliferation. These results suggest that aging impacts the colonic circadian clock moderately but markedly suppresses the rhythms of cell cycle genes and appears to uncouple the cell cycle machinery from circadian clock control. Conversely, tumorigenesis predominantly affects the rhythms of colonic circadian clocks but is not associated with uncoupling of circadian clock and cell cycle.
- MeSH
- buněčný cyklus fyziologie MeSH
- cirkadiánní hodiny fyziologie MeSH
- cirkadiánní rytmus fyziologie MeSH
- karcinogeneze * metabolismus patologie MeSH
- kolon fyziologie MeSH
- kolorektální nádory * metabolismus patologie MeSH
- myši MeSH
- nádorová transformace buněk MeSH
- proliferace buněk MeSH
- stárnutí * metabolismus patologie MeSH
- střevní sliznice * metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.
- MeSH
- buněčný cyklus fyziologie MeSH
- časové faktory MeSH
- chromozomy rostlin * metabolismus MeSH
- genom rostlinný genetika MeSH
- genomika metody MeSH
- hydroxymočovina MeSH
- metafáze MeSH
- mitotický index MeSH
- nitrobenzeny MeSH
- organothiofosforové sloučeniny MeSH
- průtoková cytometrie metody MeSH
- pufry MeSH
- Saccharum cytologie genetika MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Centromeres contain specialized nucleosomes at which histone H3 is partially replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. Previously, we identified a KINETOCHORE NULL 2 (KNL2) of Arabidopsis thaliana that is involved in the licensing of centromeres for the cenH3 recruitment. We also demonstrated that a knockout mutant for KNL2 shows mitotic and meiotic defects, slower development, reduced growth rate, and fertility. To analyze an effect of KNL2 mutation on global gene transcription of Arabidopsis, we performed RNA-sequencing experiments using seedling and flower bud tissues of knl2 and wild-type plants. The transcriptome data analysis revealed a high number of differentially expressed genes (DEGs) in knl2 plants. The set was enriched in genes involved in the regulation of the cell cycle, transcription, development, and DNA damage repair. In addition to comprehensive information regarding the effects of KNL2 mutation on the global gene expression, physiological changes in plants are also presented, which provides an integrated understanding of the critical role played by KNL2 in plant growth and development.
Tento druhý díl článku o prognostických a prediktivních markerech u NSCLC se zabývá dalšími potencionálně využitelnými geny/ proteiny mimo tyrozinkináz, které byly popsány v díle prvním. Jsou zmíněny geny spjaté s klíčovými drahami v růstu tumoru a jeho metastazování, stejně jako mechanismy hormonální, epigenetické i imunohistochemické a proteomické. Rovněž je zvlášť věnována pozornost markerům spojovanými s predikcí účinnosti imunoterapie a chemoterapie.
This second part of the article on prognostic and predictive markers in NSCLC addresses other potentially useful genes / proteins except tyrosine kinases that were described in the first part. There are mentioned genes with key pathways in tumor growth and metastasis as well as hormonal, epigenetic and immunohistochemical and proteomic mechanisms. Special attention is also paid to markers associated with the prediction of immunotherapy and chemotherapy.
- MeSH
- angiogenní proteiny fyziologie MeSH
- antigen CTLA-4 MeSH
- antigeny CD274 MeSH
- biologické markery MeSH
- biomarkery farmakologické MeSH
- buněčný cyklus fyziologie MeSH
- epigeneze genetická fyziologie MeSH
- genetické markery MeSH
- geny nádorové MeSH
- imunohistochemie MeSH
- kosti a kostní tkáň metabolismus MeSH
- lidé MeSH
- nádorové biomarkery * MeSH
- nádorové kmenové buňky MeSH
- nemalobuněčný karcinom plic * diagnóza patologie MeSH
- oprava DNA genetika MeSH
- prediktivní hodnota testů MeSH
- prognóza MeSH
- Check Tag
- lidé MeSH
T-2 toxin is a worldwide trichothecenetoxin and can cause various toxicities.T-2 toxin is involved in G1 phase arrest in several cell lines but molecular mechanism is still not clear. In present study, we used rat pituitary GH3 cells to investigate the mechanism involved in cell cycle arrest against T-2 toxin (40 nM) for 12, 24, 36 and 48 h as compared to control cells. GH3 cells showed a considerable increase in reactive oxygen species (ROS) as well as loss in mitochondrial membrane potential (△Ym) upon exposure to the T-2 toxin. Flow cytometry showed a significant time-dependent increase in percentage of apoptotic cells and gel electrophoresis showed the hallmark of apoptosis oligonucleosomal DNA fragmentation. Additionally, T-2 toxin-induced oxidative stress and DNA damage with a time-dependent significant increased expression of p53 favors the apoptotic process by the activation of caspase-3 in T-2 toxin treated cells. Cell cycle analysis by flow cytometry revealed a time-dependent increase ofG1 cell population along with the significant time-dependent up-regulation of mRNA and protein expression of p16 and p21 and significant down-regulation of cyclin D1, CDK4, and p-RB levels further verify the G1 phase arrest in GH3 cells. Morphology of GH3 cells by TEM clearly showed the damage and dysfunction to mitochondria and the cell nucleus. These findings for the first time demonstrate that T-2 toxin induces G1 phase cell cycle arrest by the involvement of p16/Rb pathway, along with ROS mediated oxidative stress and DNA damage with p53 and caspase cascade interaction, resulting in apoptosis in GH3 cells.
- MeSH
- buněčné linie MeSH
- buněčný cyklus účinky léků fyziologie MeSH
- geny p16 účinky léků fyziologie MeSH
- hypofýza účinky léků metabolismus ultrastruktura MeSH
- krysa rodu rattus MeSH
- retinoblastomový protein biosyntéza MeSH
- signální transdukce účinky léků fyziologie MeSH
- T-2 toxin toxicita MeSH
- viabilita buněk účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
