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. 2015 Nov 2;112(46):E6321–E6330. doi: 10.1073/pnas.1519388112

Fig. 3.

Fig. 3.

Centrosome amplification is not tolerated in vitro and in most tissues in vivo. (A) Breeding scheme to obtain mice containing the tamoxifen-inducible Cre (ERT-Cre), the Cre-inducible Plk4 transgene, and the Cre-inducible Centrin-GFP centrosomal marker. (B) Representative immunofluorescence image of a skin fibroblast derived from a Plk4 OE;ERT-Cre;Centrin-GFP animal treated for 48 h with or without 4-OHT. (C) Centrosome quantification in cells as described in B. Bars represent the mean of two independent experiments. Error bars indicate SEM. A minimum of 60 cells were analyzed for each condition. (D) Change in cell numbers over time in Plk4 OE skin fibroblasts in which Plk4 expression was induced (+ tamoxifen) or not (− tamoxifen). Lines represent the mean of two experiments. Error bars indicate SEM. (E) Clonogenic analysis of Plk4 OE; ERT-Cre;Centrin-GFP skin fibroblasts treated with (+) or without (−) 4-OHT. The graph shows the percentage of clones that grew in Plk4-overexpressing (+ tamoxifen) cells compared with uninduced controls (− tamoxifen). Bars represent the mean of two experiments. Error bars indicate SEM. Two plates were analyzed for each data point. (F) Fold change in Plk4 mRNA in the kidney of Plk4 OE; ERT-Cre;Centrin-GFP mice compared with control ERT-Cre;Centrin-GFP mice. Measurements were taken at 2 mo after tamoxifen treatment. Bars represent the mean and error bars indicate SEM. n = 3 mice with Plk4 OE (+) and n = 6 nontransgenic control mice (−). (G) Representative immunofluorescence image of a kidney section (a glomerulus is visible in the middle of the image) from Plk4 OE mice after tamoxifen treatment. (H) Percentage of centrosome amplification in cells expressing Centrin 1-GFP in the kidney of tamoxifen-treated Plk4-overexpressing mice (+) and nontransgenic control mice (−). Bars represent the mean, and error bars indicate SEM. n = 3 mice with Plk4 OE (+) and n = 6 nontransgenic animals (−). A minimum of 86 centrin-positive cells were counted for each animal. (I) Representative immunofluorescence image of a liver section from a Plk4 OE mouse after tamoxifen treatment. (J) Percentage of centrosome amplification within cells expressing Centrin 1-GFP in control livers vs. Plk4 OE livers. Measurements were obtained at 2 mo after tamoxifen treatment. Bars represent the mean, and error bars indicate SEM. n = 2 mice with Plk4 OE (+) and n = 3 nontransgenic mice (−). A minimum of 25 centrin-positive cells were counted for each animal. (K) Survival analysis of mouse cohorts treated with tamoxifen citrate food (as in Fig. S2A). Animals developing overt tumors are marked with a blue circle.