Fig. 5: sPIRp21 sensitizes cells to agents that alter the replication dynamics without directly generating DNA damage.
A Representative images of U2OS cells transduced with EV or sPIRp21 particles, mock treated (NT) or treated with Chk1i (Gö6976, 0.25 µM), ATRi (VE821, 1.5 µM), Wee1i (MK1775, 150 nM). 6 days after treatment, cells were fixed and stained with DAPI. B Cell number (mean ± SD) relative to EV-transduced, untreated U2OS cells. 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 Flow cytometry detection of SYTOX Green stained U2OS cells transduced with EV, sPIRp21 or sPIRp21ΔP particles, mock treated (NT) or treated with Chk1i at the indicated doses. 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 Cell number (mean ± SD) relative to the untreated RKO sample transduced with EV. 6 days after Chk1i (1.5 μM) treatment, 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). E Cell number (mean ± SD) relative to the untreated HeLa sample transduced with EV. 6 days after Chk1i (1.5 μM) treatment, 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). F Cell number (mean ± SD) relative to the untreated H1299 sample transduced with EV. 6 days after Chk1i (1.5 μM) treatment, 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). G Cell number (mean ± SD) relative to the untreated A549 sample transduced with EV. 6 days after Chk1i (2.5 μM) treatment, 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). H Cell number (mean ± SD) relative to the untreated PANC1 sample transduced with EV. 6 days after Chk1i (3 μM) treatment, 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). I Cell number (mean ± SD) relative to the untreated PEO1 sample transduced with EV. 6 days after Chk1i (2.5 μM) treatment, 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). J Model summarizing the versatility of p21: the cell death triggered by inducers of DNA damage or inhibitors of checkpoint proteins (which do not cause direct DNA damage) is enhanced by expression of sPIRp21.
