Fig. 2: In the absence of DNA damage, sPIRp21 but not sp21 reduces cell count in a manner that is dependent on PCNA.
A Representative images of U2OS cells transduced with the indicated vectors. Nuclei were visualized after DAPI staining. B Cell number (mean ± SD) of U2OS cells transduced as indicated and fixed at the indicated time points. The number of cells is expressed relative to the cell number of a sample transfected with the same vector, fixed 2 days post transduction. The total cell number in 3 wells from a 96-well plate was counted for each condition. N = 3 (Statistics: one-way ANOVA, Tukey post test). C FACS analysis of SYTOX green-stained U2OS cells 6 days after transduction as indicated in the panel. The dotted line represents the separation between the cells that stain positive or negative for SYTOX Green. On the right side of the dotted line, the numbers indicate the percentages of the cell population displaying high intensity of SYTOX green. A representative experiment is shown. N = 3. D Representative images of U2OS cells transduced as indicated in the panel. 48 h after transduction, cells were pulse-labeled with BrdU for 15 min before fixation, denaturation and incubation with an anti-BrdU specific antibody. E Quantification (mean ± SD) of BrdU positive cells 2 days after transduction. Cells were pulse-labeled as indicated in (D). At least 300 nuclei/sample were analyzed. N = 3 (Statistics: one-way ANOVA, Tukey post test). F Quantification (mean ± SD) of BrdU positive cells 6 days after transduction. Cells were pulse-labeled as indicated in (D) and quantified as in (E). G Quantification of BrdU intensity of the experiments shown in (E). Median is shown in black. At least 500 positive BrdU cells were analyzed. N = 3 (Statistics: Kruskal–Wallis, Dunn post test). H Quantification of BrdU intensity of experiments shown in (F). Median is shown in black. At least 400 positive BrdU cells were analyzed. N = 3 (Statistics: Kruskal–Wallis, Dunn post-test). I Quantification of IdU track length. A representative image of a DNA fiber is shown on the top portion of the panel. After incorporation of CldU for 10 min and IdU for 30 min, samples were processed according to the DNA fiber spreading protocol and the IdU track length was measured. Median is shown in black. ̴200 fibers/sample were analyzed. Data corresponding to the NT EV condition is shown also in Fig. 1F. N = 2 (Statistics: Kruskal–Wallis, Dunn post-test). J Quantification of origin firing frequency. A representative image of a DNA fiber containing an origin is shown on the top portion of the panel. The percentage of origin firing (mean ± SD) was determined in the same samples used in I as the relative number of origins [(red-green-red + red only fibers)/total fibers]. ̴400 fibers/samples were analyzed. Images used to quantify origins are the same as those used in Fig. 2I. N = 2 (Statistics: one-way ANOVA, Tukey post test).
