Anti aurora a aik

All three GIST cell lines (GIST882, GIST48, and GIST430) were kindly provided by Dr. JA Fletcher. The mutation status of the KIT gene in these cell lines has been previously described [43 (link)]. GIST882 is an IM-sensitive cell line with a homozygous missense mutation in exon 13 of KIT (K642E) [44 (link)]. The GIST430 line, which harbors a primary, heterozygous, inframe deletion in exon 11 and a secondary, heterozygous, missense mutation in exon 13 in KIT, and the GIST48 line, which harbors a primary, heterozygous, missense mutation in exon 11 and a secondary, heterozygous, missense mutation in exon 17 in KIT, are both relatively IM-resistant [43 (link)]. MLN8237, an AURKA-selective inhibitor, was purchased from Selleck Chemicals. It was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. Nocodazole was purchased from Sigma-Aldrich (CAS Number 31430-18-9). The following antibodies were used for immunoblotting: anti-Aurora A/AIK (#3092; 1:1000), anti-phospho-Aurora A (Thr288) (#3079; 1:500), anti-phospho-HistoneH3 (Ser10) (pHisH3) (#9701; 1:1000), anti-p53 (#2524; 1:1000), and anti-phospho-p70 S6 Kinase (#9205; 1:500), all from Cell Signaling Technology; anti-p21 (sc-817; 1:1000) and anti-DEC1 (sc-101023; 1:500), both from Santa Cruz Biotechnology; and anti-actin (ABS 24-100; 1:50000).

Western-blots were performed as previously described [8 (link)]. The primary antibodies were: anti-Aurora A/AIK (rabbit monoclonal, 1:100, Cell Signaling Technology®), anti-Phospho-Aurora A (Thr288) (C39D8) (rabbit monoclonal, Cell Signaling Technology®), anti-GAPDH (6C5) (mouse monoclonal, Merck Millipore, Overijse, Belgium), anti-CXCR4 (C-20) (goat polyclonal 1:100, Santa Cruz®), anti-Vimentin (rabbit monoclonal 1:1000, Cell Signaling®), anti-F-actin (NH3) (mouse monoclonal, 1:500, Abcam®), anti-β-actin-peroxidase (mouse monoclonal, 1:10 000, Sigma®), anti-acetylated tubulin (mouse monoclonal 1:100, Santa Cruz®), anti-α-tubulin (mouse monoclonal 1:100, Santa Cruz®), anti-CDC42 (mouse monoclonal 1:100, Santa Cruz®, TX, USA). Western blot quantification was performed using the Image J software (n = 3).

Immunofluorescence was performed as previously described [9 (link)]. The primary antibodies were: anti-Aurora A/AIK (rabbit monoclonal, 1:100, Cell Signaling Technology®), anti-Phospho-Aurora A (Thr288) (rabbit monoclonal, 1:100, Cell Signaling Technology®), anti-CXCR4 (mouse monoclonal, 1:100, Santa Cruz®, Heidelberg, Germany), anti-Ki-67 (mouse monoclonal, 1:400, BD Biosciences®, Erembodegem Belgium), anti-Human Nuclei (mouse monoclonal IgG, 1:250, Merck Millipore®). The specificity of the used primary antibodies was verified using IgG isotype controls for anti-Aurora A/AIK and anti-Phospho-Aurora A (Thr288) antibodies and secondary antibody incubation alone for other antibodies. The quantification of P-AurA-positive cells was normalized according to AurA/Hoechst double-positive cells per triplicated well for each condition using the Image J software (n = 3).