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. 2022 Oct 10;221(12):e202107134. doi: 10.1083/jcb.202107134

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© 2022 Weier et al.

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Figure 6. — LPS stimulation leads to PLK2 upregulation and untimely duplication of centrioles. (a) Left: Immunostaining of CEP170 in unsorted mature CETN2-GFP expressing BMDCs. Merged and individual images of CETN2-GFP/DAPI (green/blue) and CEP170 (red) are shown. Scale bar, 5 μm. Right: Schematic illustration of mother (red/gray) and daughter (gray) centrioles in cells that accumulate and duplicate centrosomes in G1. (b) mRNA expression levels of Plk1, Plk2, and Plk4 after LPS stimulation of immature WT BMDCs. mRNA levels were normalized to the expression of TATA-box binding protein. Graphs display mean values ± SD of three independent experiments. (c) Inhibition of PLK4 activity by Centrinone. CETN2-GFP expressing BMDCs were treated with either 250 or 500 nM Centrinone for the indicated time periods and centrosome numbers determined according to CETN2-GFP/ac-tubulin⁺ foci in mature BMDCs. Left: Immunostaining of ac-tubulin in Centrinone treated and control cells. Merged and individual channels of CETN2-GFP/DAPI (green/blue) and ac-tubulin (red) are shown. Right: Quantification of centriole numbers. Graphs display mean values ± SD of three and five independent experiments with at least N = 114 cells (treatment from day 6 on) and N = 146 cells (treatment during maturation) analyzed per experiment. (d) Upper panel: Immunostaininig of PCM in mature Plk2^−/− and control (scramble) CETN2-GFP expressing HOXB8-derived DCs. Merged and individual channels of CETN2-GFP (green) and γ-tubulin (red) are shown. Right images: Magnification of boxed regions. Scale bars, 5 μm. Lower panel: Quantification of centrosome numbers according to CETN2-GFP/γ-tubulin⁺ foci in mature (left) and immature (right) Plk2^−/− (cyan) and control (scramble, black) CETN2-GFP expressing HOXB8-derived DCs. Graphs display mean values ± SD of three independent experiments. N (mature) = 315/263/155 cells (scramble) and 295/268/130 cells (Plk2^−/−). N (immature) = 367/342/292 cells (scramble) and 367/388/356 cells (Plk2^−/−). **, P = 0.0095 (two-tailed, unpaired Student’s t test). Nuclei were counterstained with DAPI.