Fig. 5

Centrosome amplification induces dedifferentiation. a Overexpression of PLK4 using doxycycline-inducible MCF10A cell line [24] results in CA after 48 hours of doxycycline treatment. PLK4⁶⁰⁸ overexpresses a truncated form (amino acids 1-608) that contains the kinase domain but does not result in CA. Blue = DNA, yellow = overlap of pericentrin and gamma tubulin. b, c Flow cytometry analysis of CD24 and CD44 expression in MCF10A cells using PE-conjugated antibodies after 48 hours of doxycycline treatment. b Mean channel fluorescence (FL2) was normalized within cell lines (i.e. either PLK4^WT or PLK4⁶⁰⁸). c Bars represent the average ± SE of 3 independent experiments. d Overexpression of PLK4 using doxycycline-inducible RPE cell line [41] results in CA. e qRT-PCR analysis of cytokeratins 7, 18, and 19 normalized to 3 housekeeping genes (RRN18S, GAPDH, ACTB) in RPE cells. Bars represent average values of 2^-∆ΔCt from 3 independent experiments. f Tumors in the TMA with tubule formation in 2 of the 3 tumor regions examined were compared to tumors without tubule formation. Representative images demonstrate how the scoring was performed. Blue = DNA, green = pericentrin. g Representative images of normal and pleomorphic nuclei seen in RPE and MCF10A cell lines treated with doxycycline. The bar graphs demonstrate the average percent of pleomorphic nuclei in each condition from 3 independent experiments. Blue = DNA, green = pericentrin. *P < 0.05. Scale bars = 5 μm