Figure 5
MCPH1 depletion increases STIL levels at the centrosome. (a) Representatives image of centrioles in the indicated conditions. (b) Quantification of centrioles (centrin foci) in the indicated conditions. CHIR-124 is a CHK1 inhibitor. Milciclib is a CDK2 inhibitor. (c) Quantification of the percentage of cells with centrosome amplification, defined as the presence of > 4 centrioles (centrin foci). Bars represent means ± SEM from 3 independent experiments. In (b) and (c), each point represents a single cell, each independent experiment is coded by shape (e.g. circle, square, triangle), and different colors are used to denote the different drug treatments (e.g. red = no additional drug, blue = centrinone, green = CHIR-124, and yellow = milciclib). The larger outlined points indicate means of each independent experiment, with bars representings mean ± SEM from the 3 independent experiments. (d) Cells were transfected with p27 to inhibit CDK2. (e,f) Quantification of centrioles and CA (defined as greater than 4 centrioles) in p27-transfected cells. (g) Representative images of STIL immunofluorescence. (h–j) Quantification of STIL immunofluorescence at the centrosome in asynchronous cells (h), cells arrested in S phase with thymidine (i), and cells arrested in G2 phase with the CDK1 inhibitor RO-3306, 10 µM (j). Centrosomal STIL levels were determined by measuring STIL expression within a 2.5 µm radius of the center of the centrosome and normalizing to TetR for each experiment. In (e), (h), (i), and (j), each point represents a single cell, color coded by each independent experiment, with mean values for each experiment indicated by larger, outlined circles. Bars represent mean ± SEM from 3 independent experiments. *P value < 0.05. Scale bars = 5 µm. HeLa doxycycline-inducible MCPH1 shRNA cell line is used throughout this figure.
