The C-terminal half of Cep152 is required for Cep63 binding and centrosomal localisation.

<p>(A) Diagram of Cep152 full length and truncation proteins used in the following experiments. Numbers indicate amino acid positions. (B) Cep63 interacts with the C-terminal half of Cep152. MBP or MBP-Cep63 pull down experiments were carried out after incubation in lysates of 293 HEK cells expressing GFP-Cep152 full length (FL), N-terminal half (1–803), or C-terminal half (804–1654), and Cep152 proteins were detected by Western blotting with anti-Cep152 antibodies (Cep152 9AP). Input shows 10% of cell lysate used for pull down experiments. (C) The C-terminal half of Cep152 is required for its centrosomal localisation. 293 HEK cells expressing the GFP-Cep152 proteins used in (B) were stained with DAPI (blue) and anti-Centrin 3 antibodies (red), GFP direct fluorescence is shown in green. Lower panels show magnification of one centrosome (boxed region). Scale bars 5 µm.</p

Cep63-Cep152 centrosomal recruitment is downstream of Cep192.

<p>(A-D) Control RNAi or RNAi of Cep63, Cep152, or Cep192, was carried out for 4 days in U2OS cells, followed by immunofluorescence on replicate samples, with anti- γ-tubulin and anti-Cep63 (A), Cep152 (B), or Cep192 (C) antibodies. Fluorescence intensity of Cep63, Cep152, Cep192, and γ-tubulin at the centrosome were measured (graphs A-D). All intensity measurements were normalised to the mean of the control population and p values are indicated above (students’ t-test). (E-G) Images of γ-tubulin and Cep63, Cep152, or Cep192 immunofluorescence at the centrosome from the experiment shown in A-D. Scale bar 1 µm. (H) Western blots of whole cell lysates from U2OS cells used in experiments (A-G) using anti-Cep152 (Bethyl) and α-tubulin antibodies.</p

Cep63 N-terminus is required for centrosomal localisation of Cep63 and Cep152.

<p>(A) Diagram of Cep63 full length and truncation proteins used in the following experiments. Numbers indicate amino acid positions. (B) Cep63 C-terminus interacts with Cep152. Expression of Flag-Cep63 (FL), truncation proteins, or Flag-empty vector control (e) was induced in 293 FlpIn TREX cell lines by incubation with 2 µg/ml doxycycline for 72 hours, then proteins were immunoprecipitated using anti-Flag resin. Western blots show endogenous Cep152, detected by anti-Cep152 (Bethyl), and Cep63 truncations detected by anti-Cep63 (Millipore). Inputs are 5% of the lysate used for Flag IP. Red arrowhead points to Cep63 truncation 1–135 present in the Flag IP. (C-F) U2OS cells were transfected with YFP-Cep63 (FL), truncation proteins, or YFP-empty vector (e) for 48 hours. (C) Cells were stained with anti-Cep152 (red) and γ-tubulin (blue) antibodies; YFP-tagged proteins were detected by direct fluorescence (green). Scale bar 1 µm. (D) Whole cell lysates from (C) were analysed by Western blot with anti-GFP antibodies to visualise YFP-tagged proteins. (E) The localisation of YFP-Cep63 proteins to the centrosome was scored in 3 independent experiments, n >10. (F) Overexpression of Cep63 425–541 and 136–541 deplete Cep152 from the centrosome. The ratio of Cep152 to γ-tubulin fluorescence intensities at the centrosome was measured for multiple cells from the experiment shown in (C), n >10.</p

Cep63 is needed for efficient centriole duplication in human and mouse cells.

<p>(A-B) Cep63 depletion by RNA interference (RNAi) led to decreased centriole numbers in U2OS cells. Cells treated with control or Cep63 (Cep63-2) short interfering RNAs (siRNAs) for 4 days were stained with anti-centrin 2 (red) and anti-Cep63 (green) antibodies, plus DAPI (blue) to visualise DNA. Small panels show 3 times enlargements of the centrosome region with centrin 2 staining. Scale bar is 5 µm. (B) The number of centrin foci per mitotic cell was scored in three independent experiments, n>60. (C) Cep63 homozygous gene-trap mouse embryonic fibroblasts (MEFs) are devoid of Cep63 protein. Primary MEFs, at passage 3, were stained with anti-Cep63 (green) and anti-γ-tubulin antibodies (red), and DAPI (blue) to detect centrosomes and DNA, respectively. Representative images are shown of interphase and mitotic cells from two cell lines derived from littermates, one homozygous wild type (<i>Cep63^+/+</i>) and the other homozygous gene-trap (<i>Cep63^T/T</i>). Scale bars 1 µm. (D-F) <i>Cep63^T/T</i> MEFs have reduced centriole numbers. (D) Primary MEFs, passage 3, were stained with anti-centrin2 (green) and anti-γ-tubulin (red) antibodies and DAPI. Scale bars 1 µm. (E) Centrin foci were scored in mitotic cells from 3 different cell lines for each genotype at passage 3, 40Cep63^+/+ and 3 <i>Cep63^T/T</i> embryos, n >100.</p

Lack of centrosomal Cep63–Cep152 causes a delay in HsSAS-6 recruitment.

<p>(A) Cep63 is present at the PCM before HsSAS-6 recruitment. Telophase, G1 phase, S or G2 phase and mitotic HeLa cells, as indicated, were stained with anti-centrin 2 (red), Cep63 (green), and HsSAS-6 (blue) antibodies. Centrosomes from each cell are shown. Scale bar 1 µm. (B) HsSAS-6 foci were counted in U2OS cells with nuclear PCNA foci (a marker of DNA replication) after 96 hours RNAi as indicated, n >150, 3 experiments. P values from a students’ t-test are indicated on the graph. (C) U2OS cells were stained with anti-Cyclin A and HsSAS-6 antibodies after control, Cep63, or Cep152 RNAi. Cyclin A status, negative (-, early G1 phase), dull (+, G1-S), or bright (++, S-G2), and the number of HsSAS-6 foci (0,1,2,>2) were scored in asynchronous populations in three independent experiments, n >150.</p

Continuous real-time recordings of adhesion probability of VSMC to COL-I coated AFM probe following ADO or NO administration.

<p><b>(A)</b> A representative individual experiment shows the decrease in the adhesion probability after introduction of ADO in the cell bath. <b>(B)</b> Decrease in the group average adhesion probability after addition of ADO in the cell bath (n = 10). <b>(C)</b> Average adhesion probability showed a significant decrease in adhesion probability following addition of ADO. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>). <b>(D)</b> NO donor dramatically reduced adhesion probability as shown in a representative individual example. <b>(E)</b> Average adhesion events decreased after treatment with NO (n = 10). <b>(F)</b> Average adhesion probability shows a significant decrease in adhesion probability after the addition of NO donor to the cell bath. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>).</p

Continuous real-time recordings of E-modulus and adhesion probability of VSMC to COL-I coated AFM probe in sham control experiments.

<p><b>(A)</b> A representative individual example shows no effect on adhesion probability after addition of vehicle buffer. <b>(B)</b> No change in adhesion probability was shown in the group average adhesion probability before and after addition of vehicle buffer (n = 10). <b>(C)</b> Average adhesion probability summed across all time points for the group of VSMCs before and after addition of vehicle buffer (n = 10, <i>P>0.05</i>). <b>(D)</b> A representative single cell measurement shows no changes in E-modulus before and after addition of vehicle buffer in cell bath. <b>(E)</b> Group average VSMC E-modulus did not change before and after addition of vehicle buffer in cell bath (n = 10). <b>(F)</b> No significant difference was shown in the average E-modulus summed for all time points before and after addition of vehicle buffer (n = 10, <i>P>0.05</i>). Data were presented as mean ± SEM.</p

Representative force curve recorded by AFM.

<p><b>(A)</b> An example of an approach curve recorded by AFM (red). The blue diamond in the black square is the estimated contact point with the VSMC where the AFM cantilever is in contact with the cell plasma membrane and begins to bend. The blue line is the Hertz fitting to the approach curve. <b>(B)</b> Representative retraction force curve recorded by AFM for pre-drug period (control). <b>(C)</b> Representative retraction force curves after treatment with NO. <b>(D)</b> Representative retraction force curve after treatment with ANG II. The height between the paired red spots was used to compute the adhesion force and the rupture number was used to evaluate the adhesion probability. The 1.5 fold of the average noise (signal fluctuation) was set as the threshold of adhesion force and only the rupture force higher than threshold was considered as the real unbinding force between cell membrane and AFM cantilever. The rupture indicated by two green spots was considered as noise and omitted in adhesion probability computation (<b>C)</b>. The snaps with low gap height were not the particular characteristics of the NO or ADO treated force curve, they also appeared in control and ANG II treated force curve and were omitted as well.</p

Continuous real-time recordings of E-modulus and adhesion probability of VSMC to COL-I coated AFM probe following stimulation with ANG II.

<p>The adhesion probability is presented as the number of adhesion events per AFM retraction curve. <b>(A)</b> A representative individual example shows the increase in VSMC adhesion probability after stimulation with ANG II. <b>(B)</b> Average adhesion probability before and after addition of ANG II for experimental group (n = 10). <b>(C)</b> Average adhesion probability significantly increased after ANG II treatment. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>). <b>(D)</b> A representative single cell record of VSMC E-modulus shows the immediate increase in cell E-modulus after the addition of ANG II in cell bath (10⁻⁶ M). <b>(E)</b> Alteration in group average of VSMC E-modulus before and after stimulation with ANG II (n = 10). <b>(F)</b> Average E-modulus summed across all time points for the group significantly increased after addition of ANG II. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>.</p

Zymography of T-47D cancer cells following hypoxic treatment.

<p>Note the relative increased activity of LDH-5 following hypoxic growth.</p