Fig. 3: sPIRp21 increases replication stress and negatively impacts on the M phase transition in the absence of DNA damage. | Cell Death & Disease

Fig. 3: sPIRp21 increases replication stress and negatively impacts on the M phase transition in the absence of DNA damage.

From: Versatile enhancement of the killing potential of anti-cancer agents achieved by peptide mimetics of the PCNA interface towards specialized DNA polymerases

Fig. 3

A On top: Western blot of U2OS cells 48 h after transduction with EV, sp21, sPIRp21 or sPIRp21∆P. PCNA ubiquitination and PCNA were detected using specific antibodies against ubiquitinated PCNA and PCNA respectively. Ku70 was used as a loading control. A representative image is shown. Below: densitometry quantification of the bands shown above. N = 2. B On the left: Representative images showing levels of γH2AX staining in U2OS cells 48 h after the transductions indicated in the panel. UV irradiation (5 J/m2) of the EV sample was used as a positive control of the levels of induction of γH2AX by damaged DNA. On the right: representative images of single nuclei showing the distribution of γH2AX in small and big foci. C Quantification (mean ± SD) of U2OS nuclei positive for small (empty boxes) and big γH2AX foci (striped boxes). 200 cells/sample were analyzed. N = 3 (Statistics: one-way ANOVA, Tukey post test). D Representative images of U2OS nuclei positive (+) or negative (−) for EdU and positive (+) or negative (−) for big γH2AX foci (BF). White line delimits the nuclei area. DAPI channel data was used to create a mask in order to generate the white line. E Percentages (mean ± SD) of U2OS nuclei positive or negative for EdU for the population of cells with big γH2AX foci 48 h after transduction as indicated in the panels. At least 50 nuclei with big γH2AX foci/sample were analyzed. N = 3 (Statistics: one-way ANOVA, Tukey post test). F On top: Representative images of mitotic U2OS cells. Maximum intensity projection of Z-stack is shown. Bottom: percentages (mean ± SD) of mitotic cells out of U2OS cells transduced as indicated in the panel. At least 1000 cells/sample were analyzed. N = 4 (Statistics: one-way ANOVA, Tukey post test). G Representative images of U2OS nuclei positive (EdU +) or negative (EdU −) for EdU staining and positive (NB +) or negative (NB −) for 53BP1 nuclear bodies. H Percentages (mean ± SD) of EdU negative (EdU −) nuclei with 1 to 5 or more 53BP1 nuclear bodies (boxes on top) from U2OS cells 48 h after transduction as indicated in the panels. At least 150 EdU negative nuclei/sample were analyzed. N = 2 (Statistics: one-way ANOVA, Tukey post test).

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