Fig. 6: Expression of sPIRp21 potentiates the mechanism of cell killing previously reported for Chk1 inhibition.
A Representative images of GFP-Rev1 transfected U2OS cells. A positive and negative nucleus with and without focal organization of GFP-Rev1 is shown. B Percentage (mean ± SD) of U2OS cells with nuclear foci of GFP-Rev1. Samples were transfected with GFP-Rev1 and 5 h later transduced with EV, sPIRp21 or sPIRp21ΔP. 48 h later, cells were treated with Chk1i (0.5 µM) or DMSO and fixed 8 h after treatment. At least 200 nuclei positive for both GFP and myc/sample were quantified. N = 3 (Statistics: one-way ANOVA, Tukey post test). C Percentage (mean ± SD) of U2OS cells with nuclear foci of GFP-Rev1. Samples were transfected with GFP-Rev1 and 5 h later transduced with EV, sPIRp21 or sPIRp21ΔP. After 48 h, cells were treated with Chk1i (0.5 µM) or DMSO and fixed 24 h after treatment. 200 nuclei positive for both GFP and myc were analyzed/sample. N = 3 (Statistics: one-way ANOVA, Tukey post test). D Representative images showing pan-nuclear γH2AX induction in U2OS cells transduced with the indicated particles after mock treatment (NT) or Chk1i treatment (0.5 µM). E Representative images showing different nuclear organization patterns of γH2AX in U2OS cells. F Percentage (mean ± SD) of U2OS cells positive for pan-nuclear, small or big γH2AX focal organization. Samples were transduced as indicated in the panel and 24 h later treated with Chk1i (0.5 µM) and fixed 24 h after treatment. At least 200 cells/sample were analyzed. N = 3 (Statistics: one-way ANOVA, Tukey post test). G Percentage (mean ± SD) of H1299 cells positive for pan-nuclear, small or big γH2AX focal organization. Samples were transduced as indicated in the panel and 24 h later treated with Chk1i treatment (1 µM) and fixed 24 h after treatment. At least 200 cells/sample were analyzed. N = 3 (Statistics: one-way ANOVA, Tukey post test). H Percentage (mean ± SD) of RKO cells positive for pan-nuclear, small or big γH2AX focal organization. Samples were transduced as indicated in the panel and 24 h later treated with Chk1i treatment (1 µM) and fixed 24 h after treatment. At least 200 cells/sample were analyzed. N = 3 (Statistics: one-way ANOVA, Tukey post test). I Percentage (mean ± SD) of U2OS cells positive for pan-nuclear, small or big γH2AX focal organization. Samples were transduced as indicated in the panel and 24 h later treated with Chk1i (0.5 µM), roscovitine (2.5 µM) or both, and fixed 24 h later. At least 200 cells/sample were analyzed. N = 3 (Statistics: one-way ANOVA, Tukey post test). J Representative images of DAPI-stained nuclei from U2OS cells transduced with sPIRp21 and 48 h later treated with DMSO, or with Chk1i (0.5 µM), roscovitine (2.5 µM) or both for 6 days. K Cell number (mean ± SD) of experiment shown in (J). Each condition was expressed relative to the DMSO control. 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). L Cell number (mean ± SD) relative to untreated (NT) siLuc-transfected U2OS cells. Samples were transfected with siLuc or siCDC45 and 5 h later transduced with sPIRp21. 24 h later samples were mock treated (NT) or treated with Chk1i (0.5 µM). 6 days later, 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).
