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. 2016 Apr 8;7(32):50972–50985. doi: 10.18632/oncotarget.8646

Figure 4. Effects of transduced E7 in HPV-16-null C-33A CC cells.

Figure 4

The HPV16-null C-33A cells were transfected with E7 oncoprotein (E7 wt) or one of its deletion mutants E7Δ62-66 and E7Δ71-75. (A) Left panel. Bar graph showing the mean ± SD of the percentages of transfected cells (FL1 positive cells) obtained by flow cytometry in three different experiments. Right panel. Bar graph showing the mean ± SD of the percentages of transfected cells in the S phase of the cell cycle (FL1/FL4 double positive cells) obtained by flow cytometry analysis in three different experiments. (B) Flow cytometry histograms of transfected cells obtained in a representative experiment. Numbers represent the percentages of FL1 positive cells. (C) Biparametric flow cytometry analysis of the cell cycle restricted to transfected (FL1 positive) cells only. The y axis depicts the degree of BrdU fluorescence: cells actively synthesizing DNA incorporate BrdU (S phase of the cell cycle); the x axis portrays PI fluorescence. Numbers into the boxes indicate the percentage of the transfected cells in S phase of the cell cycle. Representative dot plots are shown. (D) Quantitative evaluation of GSN/E7 association by FRET technique, as revealed by flow cytometry analysis in FL1 positive cells (transfected). Numbers indicate the percentage of FL3-positive events obtained in one experiment representative of three. Inserts represent GSN intracellular amount in the corresponding sample and was quantitatively expressed by the median fluorescence intensity. (E) Left panel. Bar graph showing the FE, calculated according to the Riemann's algorithm, of GSN/E7 association. Data are reported as mean ± SD from three independent experiments. Central panel and right panel show the mean ± SD of GSN and E7 intracellular amount, respectively, obtained from three independent experiments and quantitatively expressed by the median fluorescence intensity. Dashed lines designate the median fluorescence value of a negative control (baseline). (*) Indicates P < 0.01 vs. mock-transfected cells.