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Figure S5. LI Analysis (A and B) Analyses of the BrdU-LI (the proportion of BrdU-labeled cells) in wild-type (wt) (A) and lipofected (B) retinas. BrdU was injected and detected as described [19,31]. Images in (A) show examples of retinal sections of wt embryos, after different times of BrdU in incorporation (h BrdU) starting from st. 30 and st. 34. White lines demarcate the central retinal region that was considered for quantitative analysis. BrdU-positive nuclei are detectable in green among DAPI-stained (blue) nuclei. L: lens. Images in (B) show magnifications of retinas (bordered by dashed lines) lipofected at st. 17 with cdk2/cyclinA2 (cdk/cyc [18]), or with XE2F [42], after BrdU cumulative incorporation from st. 34. White arrows point at BrdU-positive nuclei (detectable in red) of lipofected cells (detectable in green). Histograms show the percentage of BrdU-immunopositive cells among DAPI-positive cells (A), or lipofected cells (B), after cumulative BrdU incorporation; h indicates the time of incorporation (in hours); n reports the total number of cells counted; and error bars show standard error of the mean. Diagrams show the linear function used to calculate the length of the cell cycle, according to Takahashi et al. [39]; abscissae indicate time (in hours); ordinates show LI of wt (A) or lipofected (B) cells; and Tc and Ts estimate the duration in hours of cell cycle and S phase, respectively. Tc and Ts of control-lipofected cells (not shown) are virtually the same as those of st. 34 wt cells. In wt retinas, the proportion of cycling cells, expressed as 10 h LI, is higher at st. 30 (32% ± 0.34% standard error of the mean) than at st. 34 (23% ± 0.1% standard error of the mean). This proportion, at st. 34, is even higher in cdk2/cyclinA2–lipofected cells (45% ± 0.1% standard error of the mean) and in XE2F-lipofected cells (48% ± 2% standard error of the mean), which both delay the exit from the cell cycle. In wt retinas, the average cell cycle length, as evaluated by Tc value, increases from st. 30 (Tc = 5.1 ± 1.3 h) to st. 34 (Tc = 8.1 ± 0.6 h). Notably at st. 34, XE2F lipofection significantly reduces Tc (Tc = 5.5 ± 1.2 h) compared to control cells of the same age (Tc = 8.1 ± 0.6 h), whereas Tc is not significantly affected by cdk2/cyclinA2 lipofection (Tc = 7.8 ± 0.9 h). The changes in Tc observed among different types of cells are poorly affected by Ts, which ranges from 0.9 to 1.4. https://doi.org/10.1371/journal.pbio.0040272.sg005 Image published in: Decembrini S et al. (2006) Copyright: © 2006 Decembrini et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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