Figure 1: Centrosome amplification disrupts autophagy.

(A) Micrographs of fixed cells probed for autophagososomes (p62/SQSTM1, red; LC3B, white) and centrioles (centrin, green) in a doxycycline-inducible PLK4 overexpression system. The PLK4 WT cell line overexpresses full length PLK4 when treated with doxycycline, while the ΔC cell line overexpresses a truncated form of PLK4 (the first 608 amino acids, lacking the C-terminal localization domain) and does not result in centrosome amplification. The centrin panel shows enlargement of the centrosome. Scale bar = 5 μm. (B) Quantification of p62 immunofluorescence. Chloroquine, an autophagy inhibitor, was used as a positive control. (C) Quantification of p62 foci exceeding a predefined threshold in the indicated cell lines pictured in panel A. In panels B, C, and D, dots represent individual cells. To delineate individual cells and to ensure we assessed only intracellular puncta, we co-stained for α-tubulin. (D) Quantification of LC3B immunofluorescence. (E-F) RPE PLK4 WT (E) and MCF10A PLK4 WT (F) cells were treated with doxycycline and fixed after 3, 6, 12, 24, 36, and 48 hours. Centrioles (centrin foci), p62 expression, and LC3B expression were quantified. Dots represent at least 30 cells with bars indicating SD. Throughout the figure, bars represent means ± SEM from at least 3 independent experiments. *P value < 0.05 with correction for multiple comparisons.