Figure 5: KLF14 inhibits Plk4-directed centriole replication.

(a,b) KLF14 reduction induces centriole overduplication via Plk4 repression. (a) HeLa cells were infected with vector (Control) or KLF14-knockdown (Si-1 or Si-2) lentivirus for 72 h. Centrin (red) and DNA (blue) were costained with anti-centrin antibody and 4,6-diamidino-2-phenylindole (DAPI) and visualized by fluorescence microscopy (scale bar, 5 μm). Insets show magnification of the centriole area. Bar graphs showing fraction of cells with >4 centrioles per cell. More than 100 cells were counted from 3 independent experiments, **P<0.01. (b) HeLa cells were infected with indicated lentiviral constructs for 72 h. Centrioles were stained and visualized as in a. Bar graphs showing fraction of mitotic cells with >4 centrioles per cell. More than 50 cells per experimental group were counted from 3 independent experiments, **P<0.01. Representative western blotting showing knockdown efficiencies of KLF14 and Plk4. (c) KLF14-WT and -KO passage 3 (P3) MEFs were infected with vector (Control) or mouse Plk4-knockdown lentivirus for 48 h. Centrosomes and DNA were stained with γ-tubulin (red) and DAPI (blue). Bar graphs showing fraction of cells with >2 centrosomes per cell. Data represent mean±s.d. of three separate experiments, *P<0.05. Representative western blotting showing the knockdown efficiencies of Plk4. Relative protein levels of Plk4 were quantified by densitometry. (d,e) KLF14 overexpression inhibits centriole replication. (d) HeLa cells were transfected with vector (Control) or GFP-KLF14 for 48 h. Centrin (red) and DNA (blue) were stained and visualized as in a (scale bar, 5 μm). Insets show magnification of the centriole area. Bar graphs showing fraction of cells with aberrant centriole numbers in mitosis. More than 100 cells per experimental group were counted from 3 independent experiments, **P<0.01. (e) U2OS cells were transfected with green fluorescent protein (GFP) or GFP-KLF14 for 4 h followed by HU treatment for 48 h. Centriole (red) and DNA (blue) were stained and visualized as in a (scale bar, 5 μm). Insets show magnification of the centriole area. Bar graph showing percentage of cells with centriole overduplication during prolonged S-phase. More than 100 cells per experimental group were counted from 3 independent experiments, **P<0.01.