Statistical analysis of Aurora-A and pVHL levels and localization in ccRCCs according to their Fuhrman grade and <i>VHL</i> status.

<p>pVHL expression level (A) and localization (B and C) in ccRCCs were analysed relative to the Fuhrman grade of the tumours. Aurora-A expression level was assessed relative to the VHL status (D) and Fuhrman grade (E) of the tumours. Aurora-A expression according to the VHL cytoplasmic localization was also evaluated (F). The non-parametric Mann-Whitney test was used to evaluate the statistical relationships between the expression of Aurora-A and pVHL and the tumour parameters.</p

Figure 6

<p>Putative function of the interaction between VHL and Aurora-A in the ccRCC.</p

Detection of Aurora-A and VHL in healthy and tumour tissues.

<div><p>A-Western blot analysis of protein extracts from ccRCC and matching healthy tissue samples. Total proteins from tumour (T) and healthy (N) tissue samples were extracted using the RIPA buffer and analysed by western blotting with the anti-Aurora-A polyclonal antibody (1:200) and the anti-pVHL monoclonal antibody (1:500). β-actin was used as loading control.</p> <p>B-Immunohistochemical analysis of Aurora-A and VHL expression in ccRCC and matching healthy tissue samples. Tissues samples were prepared for immunohistochemistry as described in Materials and Methods and analysed using the polyclonal antibody against Aurora-A (1:100) and the monoclonal antibody against pVHL (1:200). Microphotographs were acquired with a Leica™ DMRXA microscope equipped with a CoolSnapsHQ camera (Photometrics™) (bar 10µm).</p></div

Relative expression of the Aurora-A kinase in ccRCC and matching healthy tissue samples.

<p><b>A-</b>Expression of Aurora-A in tumours and neighbouring healthy tissues. Total RNAs extracted from tumour tissues were reverse transcribed and Aurora-A expression was then analysed by PCR using specific Aurora-A primers that produced an amplicon of 150 bp. Expression values were normalized to GAPDH levels. <b>B</b>-Aurora-A transcript levels are correlated with the tumour Fuhrman grade and <i>VHL</i> status. Quantitative RT-PCR data were normalized to GAPDH and then their distribution according to the tumour grade and VHL status was analysed using the Mann-Whitney test. The Aurora-A transcription level of each tumour is symbolized by a dot and the median by a horizontal line.</p

pVHL is phosphorylated on Ser72 by Aurora-A <i>in vitro</i>.

<div><p>A- Purified recombinant 6xHis-tagged pVHL30 [VHL(His)₆] (lane 1) or mutant 6xHis-tagged pVHL30 S72A [VHLSA72(His)₆] ( (lane 2) were incubated in the presence of [γ-³²P] ATP and recombinant Aurora-A(His) 6. Samples were separated by SDS-PAGE and the gel was stained with Coomassie Blue (higher panel). pVHL phosphorylation status was analysed by autoradiography (lower panel). The asterisk (*) indicate the pVHL protein.</p> <p>B-Localization and sequence of the putative Aurora-A phosphorylation site on pVHL.</p></div

pVHL interacts with Aurora-A.

<div><p>A-HeLa cells were synchronized as described in Materials and Methods. Twenty µg of protein extracts were separated on a poly-acrylamide gel and VHL expression was assessed with the monoclonal anti-pVHL antibody (1:200). Loading was controlled with the anti-Actin antibody (1:200).</p> <p>B-In HeLa and RCC4 cells, Aurora-A and VHL co-localize at the centrosome and at the mitotic spindle extremities (anti-Aurora-A antibody, 1:200 and anti-pVHL antibody, 1:200).</p> <p>C-HeLa, RCC4 and 786-0 cell protein extracts were immunoprecipitated with the anti-pVHL polyclonal antibody (lane 2) or with the IgG (lane 3, negative control). Aurora-A and pVHL were both detected in the immunoprecipitates using a polyclonal antibody against Aurora-A (1:200) (upper panel) and a monoclonal antibody against pVHL (1:200) (lower panel). Lane 1 was corresponding to the total cell extract.</p></div