MS/MS-identification of proteins detected by SERPA from colorectal cancer cells exposed to hypoxia.

<p>Mapping of spots of interest resulting from the comparison described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076508#pone-0076508-g001" target="_blank">Fig.1</a> and list of identified proteins (p<0.001) obtained using lysates of HCT116 colorectal cancer cells.</p

Hypoxia integration in the SERPA strategy.

<p><b>A.</b> Workflow of the SERPA process including 2DE-gel separation of lysates from either hypoxic or normoxic tumor cells, membrane transfer, immunoblotting with the serum from either control or tumor-bearing mice, and detection of spots of interest. <b>B.</b> Typical immunoblotting patterns resulting from the incubation of 2D-resolved lysates of HCT116 cells exposed to normoxia or hypoxia, with the indicated mouse serum. In the bottom panels, proteins of the lysates are labelled with Cy dye (red) and fixed antibodies are detected with an anti-mouse secondary antibody (green spot); arrow indicates the presence of a protein exclusively detected in the lysates of hypoxic tumor cells by antibodies from the serum of tumor-bearing mice.</p

Validation of phospho-eEF2 protein as the target of autoantibodies in mice bearing colorectal HCT116 tumors.

<p><b>A.</b> Representative immunoblotting of 2D-separated lysates of hypoxic HCT116 cells with a commercial antibody against eEF2. Proteins of the lysates are labelled with Cy dye (red) and secondary antibody is conjugated to horseradish peroxidase (green spots). Positive signal is obtained for several spots of the same molecular weight but differing by their pI value. <b>B.</b> Comparison of the eEF2 spots detected with a commercial antibody against total eEF2 (top), the serum from tumor-bearing mice (middle) and a commercial antibody against phospho-Thr56 eEF2 (bottom). Spot 4 (rightmost spot) corresponds to the unphosphorylated form of eEF2 while the other spots correspond to multi-phosphorylated forms of the protein; spot 3 (second spot from the right) corresponds to the preferential monophosphorylated form of eEF2 (on Thr56).</p

Validation of hypoxia-induced phosphorylations of eEF2 in colorectal cancer cells.

<p><b>A.</b> Representative eEF2 and phospho-Thr56 eEF2 immunoblotting of HCT116 and HT29 cultured for 48 hours under hypoxia (Hx) or maintained in normoxia (Nx). <b>B.</b> Normalized expression of phospho-Thr56 eEF2 in normoxic vs hypoxic HCT116 and HT29 cells; n = 3, **p<0.01 <b>C.</b> Representative phospho-Thr56 eEF2 immunostaining of sections of HCT116 tumors in the absence (top) or the presence (bottom) of phosphatase lambda; note the complete disappearance of the phosphorylated form of eEF2 upon treatment with the phosphatase.</p

Changes in the titer of anti-phospho-eEF2 aAb as a marker of early tumor progression in mice and humans.

<p><b>A.</b> Human and mouse amino acid sequences of eEF2 in the region of Thr56. The 12 residues corresponding to the synthetic peptide (phosphorylated on Thr56) used in our immunoassay are indicated (red frame); note the perfect identity between mouse and human sequences. <b>B.</b> Detection of commercial anti-phospho-Thr56 eEF2-antibodies using our immunoassay; dashed lines show the 95% confidence band of the linear regression. <b>C.</b> Detection of anti-phospho-Thr56 eEF2 aAb in the serum of control or HCT116 tumor-bearing mice (n = 3). ***P<0.001. <b>D.</b> Time course of HCT116 tumor growth as determined by measurements of tumor diameters (n = 7 per group). <b>E.</b> Detection of anti-phospho-Thr56 eEF2 aAb at the indicated time of HCT116 tumor progression. *P<0.05, **P<0.01, ***P<0.001 (n = 6–7 per group). Note that at day 7 post-implantation, tumors are not detectable (see panel D) but a positive signal is detected in the immunoassay. <b>F.</b> Graph represents the detection of anti-phospho-Thr56 eEF2 aAb in the serum of control subjects (n = 6) and patients with adenomatous polyps (n = 14) or carcinoma (n = 9). *P<0.05, **P<0.01. Of note, K-means clustering identified two subpopulations of patients (see black and red symbols) among individuals diagnosed with adenomatous polyps (P<0.001) and carcinoma (P<0.01); the same partition was observed in 100 independent runs by varying the random initialization of K-means algorithm.</p