Figure 5.

Extra centrosomes are necessary and sufficient for PIDDosome activation. (A) Scheme of the protocol used to generate polyploid cells with or without extra centrosomes. A549 cells were treated with either solvent control (DMSO), ZM447439 (ZM), or nocodazole plus reversine (NocRev) to promote cytokinesis failure in the absence or presence of 24 or 48 h of centrinone treatment. The combination of nocodazole plus reversine appeared best suited for faithful enumeration of centrioles by immunofluorescence (see below). Cells treated as in A were subjected to DNA content analysis (B), immunofluorescence (C,D), and immunoblotting with the indicated antibodies (F). (D) Scatter plot of cells stained as in C subjected to counting of C-Nap1-positive centrioles per cell. Fifty cells per condition were analyzed. Black dashed lines represent the median. (E) Summary of the features measured across the indicated treatments concerning ploidy and centrosomal content. The cartoon summarizes the typical centrosomal arrangement, with blue cylinders representing centrioles, ±green triangles representing the appendage structures decorating them, and markers (as Cep164) for mother centrioles. (G,H) U2OS-Trex-MYC-PLK4 cells were left untreated or treated with doxycycline (DOX) for 72 h and subjected to immunoblotting with the indicated antibodies (G) or DNA content analysis (H).