Fig. 6. Centrosome amplification leads to PIDDosome-dependent cell death.

(A) Time-dependent caspase-3 substrate processing in multipotent hematopoietic progenitors (MPP) after doxycycline treatment was measured over time by IncuCyte analysis (red fluorescence area/phase area in μm²). rtTA (five biological replicates), rtTA/Plk4 (four biological replicates). (B) Quantification of DAPI and annexin V–positive MPP cells after 24 or 48 hours of doxycycline exposure. rtTA (two biological replicates), rtTA/Plk4 (four biological replicates), rtTA/Plk4/Casp2^−/− (four biological replicates), rtTA/Plk4/Raidd^−/− (two biological replicates), rtTA/Plk4/Pidd1^−/− (two biological replicates). Two to five technical replicates were performed. (C) Flow cytometric evaluation of DAPI and annexin V staining of MPP of the indicated genotypes after 48 hours of doxycycline exposure. pMIG retroviral expression vectors were used to transduce MPPs of the indicated genotypes with human BCL2. rtTA (n = 1), rtTA/Plk4 (two biological replicates); rtTA/Bcl2 (n = 1), rtTA/Plk4/Bcl2 (two biological replicates). Two technical replicates were performed. (D) Western blot analysis of pMIG and pMIG BCL2-transduced MPP kept 48 hours on doxycycline. Two technical replicates were performed. (E) Western blot analysis of MPP protein extracts isolated from MPPs of the indicated genotypes 36 or 72 hours after addition of doxycycline. Two technical replicates were performed. (F) qRT-PCR analysis of Plk4, (G) p21, and (H) Bax mRNA in BCL2-overexpressing MPPs of the indicated cell lines in (E), kept for 72 or 96 hours on doxycycline. Two technical replicates were performed. Data are shown as means ± SD and tested by Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.005.