Figure 4
MABs proliferation and cell cycle analysis upon IdU treatment at low seeding density. (a) Representative image of the thymidine-competition assay. MABs were treated with 25 μM IdU, 25 μM thymidine and in combination, harvested after 5 days and stained for alkaline phosphatase activity. Scale bar 200 μm. (b) ALP positive area quantitation of images in panel (a). Data are presented as percentage of ALP positive area over the total area of the image filed ± SEM. Statistical significance was assessed by a Two-Way ANOVA test. ***p < 0.001; ##p < 0.01; n = 3. (c) Quantitation of MABs nuclei stained with Hoechst 33342 in control cultures and in cultures treated with 25 μM IdU. Data are presented as mean of nuclei per field ± SEM. Statistical significance was assessed by a Two-Way Anova test taking Vehicle treated sample as reference. ****p < 0.0001; n = 3. (d) Exponential growth analysis of data in panel (c). The doubling time of Vehicle and IdU treated MABs was analyzed with Non-linear regression and Exponential growth equation tool of Graph Pad Prism 6. Doubling time is presented as mean ± SEM. The statistical significance between the two doubling time data sets was assessed by a Student’s t-test. **p < 0.01. n = 3 (e) Analysis of single cell DNA content as measured by cytofluorimetry of propidium iodide stained cells. 10,000 cell events were analyzed for each sample. Data are presented as counts over FL2-A fluorescence. Diploid nuclei in blue. S phase in green. Tetraploid nuclei in red. (f) Quantitation of cell cycle analysis. Data are presented as percentage of events ± SEM. Statistical significance was assessed by a Two-Way Anova test. *p < 0.05; n = 3. (g) Western blot for p21 of 25 μM IdU treated MABs at time zero (T0) and 24 hours after treatment. Vinculin was taken as loading control. The complete gel is reported in Figure S8. (h) Densitometric quantitation of p21 in panel (g). Values were normalized over vinculin and the fold induction was calculated over the time zero (T0 = 1) sample. Data are represented as mean ± SEM. Statistical significance was assessed by a Student’s t-test. **p < 0.01. n = 3.
