Data represented the mean ± SEM, from three independent experiments. The relative (S+G2)/G1 ratio was calculated considering NI cells at 0 hpr as a value of 1. (E) Comparison plot of relative (S+G2)/G1 ratio obtained from flow cytometry of fluorescence intensities of Ctd1-mKO2 and Geminin-mAG (gray bars) or DNA content of PI fluorescence intensity (orange bars). (D) Interphase stages plot (early/late G1, G1/S and S/G2/M). The data were obtained using DJF model where purple, yellow and green areas under the curve correspond to the values of G1, S and G2 phases, respectively. (C) DNA content histograms determined by PI fluorescence intensity. (B) Density plots.The cells were discriminated by its Ctd1-mKO2 (red) and Geminin-mAG (green) fluorescence intensities and gated as early G1, late G1, G1/S and S/G2/M. The red, yellow and green arrowheads indicate the cells in early/late G1, G1/S and S/G2/M phases, respectively. Each image corresponds to the fluorescence merge from Ctd1-mKO2 (red), Geminin-mAG (green) and a bright field. Characterization of synchronized MA104-Fucci cells at 0, 4 and 8 hpr from thymidine. S4 Fig: Characterization of MA104-Fucci cells. (E) Images of electron microscopy of negatively stained OSU-TLPs after inactivation with UV-psoralen (UV/AMT). The molecular weight markers are indicated. Cyclin B1, cdc2-P (Tyr 15) and NSP5 were detected using specific antibodies. The cells were harvested at 0, 2, 4, 6 and 8 hpr. (D) Immunoblotting of cell lysates from OSU-infected (lanes 1 to 5) and non-infected (lanes 6 to10) synchronized Caco-2 cells. (C) Plot showing the percentage of the interphase stages (G1, S, and G2) from synchronized non-infected (NI) and OSU-infected Caco-2 cells at the indicated times post-release from thymidine. Each histogram overlays the DNA content by DJF mathematical model where purple, yellow and green areas under the curve correspond to the values of G1, S and G2 phases, respectively. (B) Flow cytometer histograms of synchronized Caco-2 cells (Human colon adenocarcinoma cells) infected with porcine OSU strain and analyzed at 0, 2, 4, 6 and 8 hpr from thymidine.
The merged image is presented in the right column.
Cells were fixed in paraformaldehyde and immunostained for cyclin B1 (mouse mAb anti-cyclin B1, red) or PCNA (mouse mAb anti-PCNA, red), viroplasms detected with NSP5-EGFP (green) and nuclei stained with DAPI (blue). (A) Immunofluorescence of RRV-infected synchronized NSP5-EGFP/MA104 cells at 6 hpr. S2 Fig: Detection of cyclin B1 and PCNA in RRV-infected cells, RV-infected synchronized Caco-2 cells and EM of inactivated RV virions.
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