IHC and FISH analysis of AKT2 in NSCLCs.

<p>A, left: SCC negative for AKT2 expression; right: SCC positive for AKT2 expression. B, left: ADC negative for AKT2 expression; right: ADC positive for AKT2 expression. Magnification 10× and 40×, respectively. C. Dual-colour fluorescence in situ hybridization analysis of AKT2 gene copy number. FISH analysis of AKT2 (red signals) and chromosome region 19p13.1 (green signals). Left, NSCLC sample with diploid cells; right, NSCLC sample with multiple clustered spots of red signals of AKT2 with 2 chromosome region 19p13.1 signals (gene amplification). Original magnification 100×.</p

Correlation between AKT activation and clinico-pathological features of NSCLC patients.

a<p>AKT activation was evaluated with phospho-specific antibodies (pS473) and scored as negative (<10% of the tumour cells with weak, focal immunopositivity or absence of staining) and high (>10% of tumour cells with strong or diffuse immunopositivity).</p>§<p>G1–G2 vs G3–G4.</p><p>*Stage II vs Stage III.</p

The top 11 canonical signalling pathways influenced by constitutive PI3K signaling.

<p>Active PIK3CA (E545K)-expressing lentivirus was transduced in non-transformed lung epithelial cells (BEAS-2B). Transduced cells were selected in blasticydin, checked for expression of the exogenous PIK3CA-E545K, were analysed for their transcriptomes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030427#s2" target="_blank">Materials and methods</a>. <b>A.</b> Immunoblot for PIK3CA expression in transfected BEAS-2B cells. <b>B.</b> Heat map showing fold change patterns of DEGs induced by constitutive PI3K signalling. The heat map was generated in Matlab (Mathworks), and compares fold change patterns of DEGs in BEAS-2B-PI3K-E545K cells compared to parental BEAS-2B (p<0.01). Red: up-regulated genes; green: down-regulated genes. Fold changes of all down-regulated DEGs and all but one up-regulated DEG are #8 (central color spectrum bar). <b>C.</b> The top 11 functional categories determined by IPA, that were significantly up-regulated or down-regulated in BEAS-2B-PI3K-E545K cells compared to parental BEAS-2B are shown. The 2126 DEGs in BEAS-2B-PI3K-E545K were mapped to the IPA-defined network. The significance p-values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log (p-value). <b>D.</b> Bio-functions identified by IPA in the 2126 DEGs from BEAS-2B-PI3K-E545K compared with BEAS-2B. <b>E.</b> Sub-Categories and Functions identified through IPA showing the genes associated to lung cancer in the 2126 DEGs from BEAS-2B-PI3K-E545K compared with BEAS-2B.</p

Correlation between AKT activation (pAKT) and clinico-pathologic features of patients with OC.

a<p>Patients for which pAKT staining was available (N = 93).</p>b<p>Patients for which both Grade and pAKT staining were available (N = 87).</p>c<p>Patients for which both Figo Stage and pAKT staining were available (N = 93).</p><p><b>NS</b>: not significant.</p

pS473 AKT immunostaining (IHC) in NSCLCs.

<p>A, left: SCC negative for pAKT phosphorylation; right: SCC positive for pS473 phosphorylation. B, left: ADC negative for pAKT phosphorylation; right: ADC positive for pS473 phosphorylation. Magnification 10× and 40×, respectively.</p

Immunostaining analysis of PIK3CA in OC.

<p>A. Left: S-OC negative for PIK3CA expression; right: S-OC positive for PIK3CA expression. B. Left: E-OC negative for PIK3CA expression; right: E-OC positive for PIK3CA expression. Magnification 40X. Magnification of the insets 10X.</p

Correlation between AKT activation and the presence of genetic alterations in PIK3CA, AKT1 and AKT2 in OC.

a<p>Low polisomy and negative samples for which pAKT staining was available.</p>b<p>High polisomy and amplified samples for which pAKT staining was available.</p

Correlation between AKT activation and expression of the different members of the PI3K pathway in NSCLCs.

a<p>AKT activation was evaluated with as pS473 positivity and scored as negative (<10% of positive tumour cells) and high (>10% of positive tumour cells).</p>b<p>PIK3CA was graded as positive (>25% of tumour cells showed strong or diffuse immunopositivity) as moderate (>10% of tumour cells showed moderate immunopositivity) or negative (0–10% of the tumour cells showed weak, focal immunopositivity or absence of staining).</p>c<p>PTEN expression was classified as (+) when staining was detected in >50% of the cells, (+/−) when staining was detected in 25–50% of cells and (−) when staining was detected in 0–25% of cells. For statistical analysis PTEN expression was considered lost when samples were classified as (−).</p>d<p>AKT1 was graded as positive (>25% of positive tumour cells) as moderate (>10% of of positive tumour cells) or negative (0–10% of positive tumour cells).</p>e<p>AKT2 was graded as positive (>25% of positive tumour cells) as moderate (>10% of positive tumour cells) or negative (0–10% of positive tumour cells).</p>§<p>Statistically significant.</p

AKT activation in OC.

a<p>Patients for which pAKT staining was available (N°).</p>b<p>Normal vs Tumour Tissue.</p><p><b>S-OC: S</b>erous <b>O</b>varian <b>C</b>arcinoma.</p><p><b>E-OC: E</b>ndometrioid <b>O</b>varian <b>C</b>arcinoma.</p><p><b>CC-OC: C</b>lear <b>C</b>ell <b>O</b>varian <b>C</b>arcinoma.</p><p><b>Mu-OC: M</b>ucinous <b>O</b>varian <b>C</b>arcinoma.</p><p><b>M-OC: M</b>ixed <b>O</b>varian <b>C</b>arcinoma.</p><p><b>NS</b>: not significant.</p

Immunostaining and gene copy number analysis of PTEN in OC.

<p>A. Left: S-OC negative for PTEN expression; right: S-OC positive for PTEN expression. B. Left: E-OC negative for PTEN expression; right: E-OC positive for PTEN expression. Magnification 40X. Magnification of the insets 10X. C. Q-RT PCR of PTEN mRNA expression in normal ovarian tissues and OC. D. Q-PCR analysis of PTEN gene copy number in normal ovarian tissues and OC. DNA from peripheral blood leukocytes (PBL) was used as reference. PTEN copy number in PBL was set arbitrarily as 2.</p