Physical characterization of cells in mock, 20 mM HU and 0.06% MMS treated cultures.

Abstract

<p>A G1 cell population was obtained by centrifugal elutriation and propagated for 1 h (mid-S phase) prior to the addition of HU or MMS. (A) Cell density analysis by direct counting on a hemocytometer). (B) Visualization of the cytokinetic furrow (a marker for cell division) in fixed DAPI-stained cells. Approximately 40% of mock-treated cells generated a cytokinetic furrow 2 h after G1 phase isolation (1 h after HU or MMS addition in treated samples). T = 4 h, mock versus HU: (student T test) two-paired P value 0.0002); T = 4 h, mock versus MMS: (student T test) two-paired P value <0.0001). (C) Plot of DNA content (PI, propidium iodide) in mock and HU-treated cells as a function of exposure time. Elutriated G1 phase cells were treated with 20 mM HU beginning 1 h after isolation (vertical arrow, mid-S phase addition) and samples were collected every 30 min for 4.5 h. 30,000 cells were scored at each time point. (D) Analysis of flow cytometry side scatter (SSC) in mock and HU-treated cells. HU was added during S phase and samples were collected at 1 h intervals and subjected to statistical analysis. The results of three independent experiments (n = 3) are compiled. (E) Visual representation of flow cytometry side scatter (SSC) and forward scatter (FSC) in cells synchronized by centrifugal elutriation. Controls: mock-treated G1 cells (T = 0 h after elutriation), mock-treated S phase cells (T = 1 h after elutriation), and mock-treated cells 6 h after elutriation. Experimental samples: 6 h HU exposure beginning in G1 phase (0 h after elutriation; (HU/G1, 6 h), or in S phase (1 h after elutriation (HU/S, 6 h). The higher complexity seen in HU-treated cells was not observed at any stage of the cell cycle in untreated controls (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005405#pgen.1005405.s003" target="_blank">S3 Fig</a>). (F) Left panel: the total cell area for 100 microscopic cell images was measured in mock S phase and HU-treated cells. The average area was determined and subjected to statistical analysis. A 30% increase in cell size was observed in HU-treated cells (T = 8 h HU versus mock S phase). Student T test two-paired P value <0.0001 (highly significant). Right panel: representative micrograph illustrating the size difference between mock-treated S phase cells and S phase cells arrested with HU for 8 h. (G) Total macronuclear area for 100 microscopic cell images was measured in mock and HU-treated cells. The average area was determined and subjected to statistical analysis. A 20% decrease in the size of the macronucleus was observed in HU-treated cells (T = 6 h HU versus mock G1 phase cells). Student T test two-paired P value <0.1 (significant). (H) Production of macronuclear extrusion bodies. DAPI staining was used to identify cells with DNA masses that were not associated with the micro- or macronucleus (macronuclear extrusion bodies). T = 4 h, mock versus HU. Student T test, two-paired P value: 0.2249 (not significant).</p

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Last time updated on 12/02/2018

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