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. 2016 Feb 26;15(8):1034–1045. doi: 10.1080/15384101.2016.1152429

Figure 3.

Figure 3.

Growth of Flp-In NIH-3T3 cells is altered by single-copy Jade-1S insertion. A) The mutant Jade-1S S18/20A.GFP cell line displayed an elongated cell shape as cells approached confluency. If allowed to grow until super-confluent, Jade-1S S18/20A.GFP cells displayed increased membrane spreading, and nuclei did not achieve close proximity as was seen in the other cells. Scale = 50 µm. B) Cells were seeded at 200 000 per 10 cm culture dish and counted 48h and 96h later. Fold change from 48h to 96h is shown. The Jade-1S S18/20A.GFP line proliferated significantly more slowly than the control GFP.GFP line (F(2,4) = 8.553, p = 0.0359; Tukey's post hoc, * = p <0.05). C) Representative histograms of propidium iodide (PtdIns) staining of each Flp-In cell line. Cells were seeded at 2 × 10e6 /10 cm dish and harvest after 24 hours for flow cytometry. D, E, F) Quantification of flow cytometry data where the first histogram peak corresponds to cells in G1/G0 phase (2n DNA) and the second peak to G2/M (4n DNA), with S phase corresponding to cells with an intermediary DNA status. The percentage of cells in cell cycle phases G1/G0 (D), S (E), and G2/M (F) was calculated for each Flp-In cell line using Cyflogic software. Results were analyzed using repeated measures ANOVA to maintain integrity of individual experimental repetitions. The wild-type Jade-1S.GFP and the mutant Jade-1S S18/20A.GFP cell lines had a significantly increased percentage of cells in G1/G0 (D) (F(2,4) = 13.77, p = 0.0161; Tukey's post hoc *, p < 0.05; **, p < 0.01). A significantly smaller percentage of wild-type Jade-1S expressing cells were in S-phase compared to the control GFP.GFP expressing cells (E) (F(2,4) = 11.15, p = 0.0231; Tukey's post hoc *, p<0.05). The mutant Jade-1S S18/20A.GFP cell line had a significantly lower percentage of cells in G2/M than the GFP.GFP control line (F) (F(2,4) = 16.97, p = 0.0111; Tukey's post hoc **, p < 0.01). G) The ratio of percentage cells in G2/M to S-phase was significantly increased in cells expressing Jade-1S S18/20A.GFP (F(2,4) = 8.497, p = 0.0363; Tukey's post hoc *, p < 0.05). Errors bars represent SEM.