Nuclear localization of CDK5RAP3 was important for the suppression of p14ARF promoter activity.

<p>(<b>a</b>) <i>S</i>chematic diagram of CDK5RAP3 mutants (<b>b</b>) Western blotting showing the expression levels of CDK5RAP3 mutants overexpressed in HepG2. Protein lysates from reporter assay were used for Western blotting probed with anti-Myc antibody. (<b>c</b>) <i>D</i>ual luciferase reporter was performed by co-transfection of CDK5RAP3 mutants with p14^ARF-luc reporter in HepG2. Results were mean of three independent experiments, with promoter activity of vector control set as 100%. *, <i>P</i><0.05 and **, <i>P</i><0.005 compared with vector control, Student’s <i>t</i>-test. (<b>d</b>) Confocal images of wild type (WT) and the indicated deletion mutants of Myc-CDK5RAP3.</p

Regulation of p14^ARF localization and protein expression by CDK5RAP3.

<p>(<b>a</b>) The p14^ARF and CDK5RAP3 protein levels in stable CDK5RAP3 knockdown SMMC-7721 clones (shCDK5RAP3#1 and #2), vector control and parental cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042210#pone.0042210-Mak1" target="_blank">[5]</a> were compared by Western blotting using indicated antibodies, respectively. (<b>b</b>) The stable CDK5RAP3 knockdown SMMC-7721 (shCDK5RAP3#2) and vector control cells were treated with 100 µg/ml cycloheximide or DMSO (vehicle), and harvested at the indicated time points. p14^ARF and <i>β</i>-actin protein levels were determined by Western blotting. V: DMSO treatment. <i>Top</i>: Western blotting; <i>bottom</i>: quantification of p14^ARF protein level.</p

Suppression of endogenous expression of p14^ARF by CDK5RAP3.

<p>(<b>a</b>) The <i>p14^ARF</i> and <i>CDK5RAP3</i> mRNA expression in stable CDK5RAP3 knockdown SMMC-7721 stable clones was determined by Quantitative real-time PCR (qPCR). Data was analyzed by comparative Ct method. Band intensity was analyzed using AlphaEasePC software and normalized with <i>β-actin</i>. Results were mean of three independent experiments. *, <i>P</i><0.005, Student’s <i>t</i>-test. (b) Similar to (a), the CDK5RAP3 stable expressing HepG2 clones (CDK5RAP3#1 and #2), vector control and parental cells were used for qPCR assay. Results were mean of three independent experiments. *<i>P</i><0.04 and **<i>P</i><0.02 compared with vector control, Student’s <i>t</i>-test. (c) The CDK5RAP3 expression construct and p14^ARF luciferase reporter, pGL3-p14^ARF were co-transfected into SMMC-7721 cells for dual-luciferase reporter assay. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.05 compared with vector control, Student’s <i>t</i>-test. (d) Similar to (c), luciferase reporters carrying truncation mutants of the p14^ARF promoter, CDK5RAP3 expression construct (0.3 µg) and vector (0.3 µg) were used for dual-luciferase reporter assay. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.02 compared with vector control, Student’s <i>t</i>-test. (e) CDK5RAP3 bound <i>p14^ARF</i> promoter by performing chromatin immunoprecipitation (ChIP) analysis on CDK5RAP3 stable overexpression clone #2 HepG2 cells (1×10⁷). Input (IN) and no antibody control (No Ab) were included. Error bars: mean ±SD.</p

Knockdown of p14^ARF reversed the suppression of cell migration and invasiveness in CDK5RAP3 knockdown HCC cells.

<p>(<b>a</b>) The CDK5RAP3 stable knockdown SMMC-7721 cells were transfected with p14^ARF or control siRNA. <i>Top,</i> Western blotting showing p14^ARF knockdown in cells; <i>bottom,</i> The bar chart showed the quantitation of migrated cells in three independent experiment (*, <i>P</i> = 0.005, Student’s <i>t</i>-test). Representative photomicrographs were shown. (<b>b</b>) Similar to <b>a</b>), but invasion assay were performed. The bar chart showed the quantitation of the invaded cells in three independent experiment (*, <i>P</i> = 0.05, Student’s <i>t</i>-test).</p

CDK5RAP3 transcriptionally regulated p14^ARF in a E2F1 independent manner.

<p>(<b>a</b>) CDK5RAP3 was co-transfected with p53-responsive element reporter for luciferase assay in HepG2 cells. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.05, **, <i>P</i><0.005 compared with vector control, Student’s <i>t-</i>test. (<b>b</b>) Expression constructs of CDK5RAP3 was co-transfected with HA-E2F1 and p14^ARF-luc reporter for luciferase assay in HepG2 cells. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.005 compared with vector control, Student’s <i>t</i>-test.</p

Rapid Discovery of Pyrido[3,4‑<i>d</i>]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach

Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido­[3,4-<i>d</i>]­pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model