CDK2 inhibition blocks CDC25B-induced centrin 2 foci formation.

<p>(A) Asynchronous U2OS-HA-CDC25B cells were incubated in the presence (tet +) or absence (tet -) of tetracycline with either 9µM CDK1 inhibitor RO-3306 [26] or 1µM CDK2 inhibitor PHA533533 [25] for 24 hours. (A) Graph represents the percentages of cells with excess centrin 2 foci. Data shown are the means of at least 200 cells counted from three independent experiments +/- SD. (B) Western blot analyses on soluble protein extracts from cells treated as in (A). Membranes were probed for HA-tagged CDC25B, centrin 2, inactive CDK (CDK-pY15P), total CDK2-cyclin E and its substrate nucleophosmin (NPM), which is phosphorylated by CDK2-cyclin E on T199. α-tubulin was used as loading control.</p

CDC25B-induced centrin 2 foci have the capacity to function as microtubule organising centres.

<p>Microtubule re-growth assay on asynchronous U2OS-HA-CDC25B cells, which were incubated in the absence of tetracycline for 5 days, and then either fixed (Control), or incubated on ice for 30 minutes, and then fixed (Cold Shock) or incubated in warm media for 1 minute (Re-growth) to allow re-assembly of microtubules. Cells were co-stained for centrin 2 (red), and α-tubulin (green), and DNA co-stained with DAPI (blue). Magnifications of the centrosomes/ centrin foci (indicated by white boxes) are shown on the right hand side. Bar = 10 µm.</p

CDC25B protects centrosomal centrin from proteasome-mediated degradation.

<p>HeLa cells were either mock-transfected or treated with scrambled control or CDC25B siRNA duplexes for 44 hours followed by 4 hour treatment with 20µM MG132. (A) Examples of cells treated (+) or not (-) with MG132, and stained for CDC25B (CDC25B-S230P, red) and centrin 2 (green). Bar = 10 µm. (B) Percentage of cells treated with control or CDC25B siRNA duplexes in which centrin 2 and CDC25B-S230P (CDC25B siRNA only) were not detectable or substantially diminished at the centrosomes. Bars represent means of at least 200 cells counted from three independent experiments +/- SD. (C) Western blots of siRNA-depleted cells treated with (+) or without (-) MG132 and immunoblotted for total CDC25B, CDC25B-S230P, γ-tubulin and α-tubulin.</p

HA-CDC25B overexpression induces the formation of extra-numerary centrin 2 “foci”.

<p>Asynchronous U2OS-HA-CDC25B cells were incubated in the continued presence (+ tet) or absence (- tet) of tetracycline as in Figure 1, fixed 48 hours later and co-stained for HA-CDC25B (red), centrin 2 (green) and DAPI (blue). (A) Examples of centrin 2 foci in cells expressing (- tet, arrows) HA-CDC25B in comparison to control cells (+ tet). Bar = 10 µm. (B) Histogram plot showing the percentage of cells harbouring excess centrin 2 “foci” following 48 hours HA-CDC25B expression. Bars represent means of at least 200 cells counted from three independent experiments +/- SD.</p

HA-CDC25B overexpression increases the level of centrin 2 at the centrosome.

<p>Asynchronous U2OS-HA-CDC25B cells were incubated in the continued presence (+tet) or absence (-tet) of tetracycline, fixed 24 hours later and co-stained for HA-CDC25B (red), centrin 2 (green) and DAPI (blue). (A) Examples of centrin fluorescence levels at the centrosome of cells expressing (-tet, arrow) or not (+tet) HA-CDC25B. Bar = 10 µm. (B, C) Analyses of centrin 2 fluorescence levels in cells expressing (-tet) or not (+tet) HA-CDC25B. (B) Cells were co-stained for centrin 2 (green) and Nek2 (red) as a marker for centrosomes. Insets are magnifications of centrosome regions (indicated by white boxes). Circles represent centrosome boundaries used to quantitate fluorescence intensities of centrin 2 and Nek2 at the centrosomes (C), using MetaMorph imaging software. (C) Box plots of the ratio of centrin 2 fluorescence intensity / Nek2 fluorescence intensity at the centrosomes of cells expressing or not HA-CDC25B. G1/S phase cells, identified as cells with a single centrosome (B, +tet) or duplicating centrosomes that were not yet separated (B, -tet), or G2 phase cells, identified as cells with 2 centrosomes that were separated, were all imaged on the same day using identical microscope and software settings. Box plots were prepared using the Prism software package and statistical analyses performed using unpaired student’s t-tests (**** P<0.0001; ns not significant, n=76-170 centrosomes). Data shown are from 1 experiment and are representative of those obtained from four independent experiments. (D) Western blot analyses of HA-CDC25B and centrin 2 levels in cells expressing or not HA-CDC25B. α-tubulin was used as a loading control.</p

CDC25B-induced centrin foci are dependent on the activity of Mps1 kinase.

<p>Asynchronous U2OS-HA-CDC25B cells were transfected with pEGFP or pEGFP-tagged wild-type (Mps1 WT), kinase-dead (Mps1 KD) or non-degradable (Mps1 Δ12/13) forms of Mps1 and incubated in the continued presence (+ tet) or absence (- tet) of tetracycline, fixed 48 hours later and co-stained for centrin 2 (red) and DAPI (blue). (A) Examples of transfected cells cultured in the absence of tetracycline. Insets are magnifications of centrosome regions (indicated by white boxes) of the transfected cell in each case. Bar = 10 µm. (B) Histogram plot showing the percentage of cells harbouring excess centrin 2 “foci” following 48 hours transfection and HA-CDC25B expression. Bars represent means of at least 200 cells counted from four independent experiments +/- SD. (C) Model for the role of CDC25B in regulating centrin 2 protein stability through its activity towards CDK2. </p

CDC25B-induced centrin 2 foci accumulate other PCM components.

<p>Asynchronous U2OS-HA-CDC25B cells were incubated in the absence of tetracycline for 48-72 hours and then fixed and co-stained for centrin 2 (red), DAPI (blue) and either Ninein (A), Pericentrin (B), γ-tubulin (C), Nedd1 (D), PCM-1 (E) or polyglutamylated (polyglut.) tubulin (F), as indicated. Images were taken using an Appotome microscope, except for C, lower panel, which is a maximum intensity projection of a z-stack taken using a laser scanning confocal microscope, to enhance the detection of γ-tubulin at distant foci. Bar = 10 µm.</p

Library clone insert size and GFP fluorescence in virus-making cells.

<p>A: scatter-plot of cloned insert size and total well GFP fluorescence in transfected HEK293T cells. B: frequency distribution histogram of clones in different insert size category.</p

Diagram summarising the key library construction steps.

<p>1. Gateway cloning was used to transfer ORFs into the lentiviral expression plasmid plv411G downstream of the EF1α promoter and upstream of the IRES driven GFP; 2. Virus production was performed by transfecting expression clones together with viral packaging plasmids into HEK293T cell line; 3. Viral supernatant was collected into a fresh set of plates and stored; 4. Transfected cells remaining in the plates were fixed and scanned to determine GFP fluorescence in each well. Frequency distribution histogram illustrates number of wells (y-axis) that had similar GFP fluorescence intensity (x-axis). Values between plates were normalized by zeroing on the mean of 4 mock transfected wells on each plate. Each plate also contained 4 empty expression vector wells, shown in green, which were used as positive controls; 5. Thirty eight ORF-expressing wells were randomly selected from each of the tail ends of the frequency distribution categories, to evaluate the performance of the viral supernatants on a range of target cell lines.</p

Transduction rates observed with low and high titer virus-producing expression clones.

<p>A,C – low titer clones; B,D –high titer clones. The bars in the inset in graph A represent mean and standard deviation (error bars) of four wells transduced with empty vector virus. Color of bars corresponds to the target cell line as indicated in the inset graph. Actively dividing human epithelial tumor (MCF7, PMC42-ET, MDA-MB-468, WMM1175), and non-tumor (MCF10A, HaCaT) cell lines were compared to non-dividing cells (WMM1175-p16, arrested by induced overexpression of p16, and primary mouse bone marrow macrophages (mBMM)). The proportion of GFP positive cells (y-axis) was determined after high-content imaging of plates containing fixed transduced cells. Each bar in A and B represents data for a well transduced with a single gene-expressing vector as indicated on x-axis. Bars representing mean values for vector wells are included in all three graphs to allow for scale comparison. Each bar in C and D represents a mean (error bar  = SD) transduction rate for all human cell lines for a given gene.</p