Fig 1: The APC/C is a component of the mitotic centrosome. (A) Mitotic RPE1 or HEK293 cells were pre-extracted, fixed in formaldehyde (top) or ice-cold methanol (bottom) and stained with the indicated antibodies. Boxes indicate regions shown in enlarged images. A line scan with a 10-pixel-wide line was performed at the indicated positions, and the normalized values were plotted (right). Data shown are representative of three experiments. (B) HEK293 cells were treated with STLC or nocodazole to induce a prometaphase arrest. Cells were pre-extracted, fixed in formaldehyde and stained with the indicated antibodies (CNT3, centrin-3). Boxes indicate regions shown in enlarged images. The intensity of APC2 at the centrosome was measured inside the dotted circle and normalized against the signal intensity in the presence of STLC (right). N cells=91 (STLC) and 84 (nocodazole). (C) HEK293 cells were treated with centrinone to deplete their centrosomes. Cells were pre-extracted, fixed in formaldehyde and stained with the indicated antibodies. The intensity of APC2 throughout the whole cell was measured and normalized against the signal intensity in DMSO (bottom). N cells=11 (DMSO control) and 14 (centrinone). In A–C, microtubules were labelled using an a-tubulin antibody, and DNA was stained with DAPI. (D) Centrosomes were purified from mitotic HEK293 cells using a sucrose gradient (centr., centrosomal). The eluted fractions were immunoblotted against the indicated proteins. The top shows PCNT, ?Tub, and centrin-1 (CNT1) as centrosomal proteins, the middle shows several APC/C components, and MCC proteins are shown at the bottom. Blots are representative of four experiments. (E) Centrosomes were purified in parallel from interphase HEK293 cells and mitotic HEK293 cells using a sucrose gradient. The first elution fractions were blotted to detect PCNT as a centrosomal marker and APC3. Blots are representative of three experiments. (F) Centrosomes from E were fixed on a coverglass and stained with the indicated antibodies (top). The intensity of APC2 was measured as indicated in B and normalized against the signal intensity on interphase centrosomes (bottom). N centrosomes=142 (interphase) and 123 (mitotic). Box plots show the median (line), 25–75% range (box) and 5–95% range (whiskers). ****P<0.0001 (Mann–Whitney U-test). Scale bars: 5 µm in A–C, 1 µm in F.