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1.
Figure 2

Figure 2. Immunostaining analysis of AKT1 in OC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Left: S-OC negative for AKT1 expression; right: S-OC positive for AKT1 expression. B. Left: E-OC negative for AKT1 expression; right: E-OC positive for AKT1 expression. Magnification 40X. Magnification of the insets 10X.

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
2.
Figure 4

Figure 4. Immunostaining analysis of PIK3CA in OC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

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.

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
3.
Figure 3

Figure 3. Immunostaining analysis of AKT2 in OC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Left: S-OC negative for AKT2 expression; right: S-OC positive for AKT2 expression. B. Left: E-OC negative for AKT2 expression; right: E-OC positive for AKT2 expression. Magnification 40X. Magnification of the insets 10X.

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
4.
Figure 1

Figure 1. AKT pS473 immunostaining analysis in OC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Left: S-OC negative for pAKT phosphorylation; right: S-OC positive for pS473 phosphorylation. B. Left: E-OC negative for pAKT phosphorylation; right: E-OC positive for pS473 phosphorylation with apical enhancement. Magnification 40X. Magnification in the insets 10X.

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
5.
Figure 6

Figure 6. Analysis of the expression and of the gene copy number of PIK3R1.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Q-PCR analysis of copy number of the PIK3R1 gene in normal ovarian tissue and OC. DNA from peripheral blood leukocytes (PBL) was used as control. PIK3R1 copy number in PBL was arbitrarly set as 2 (diploid value). B. PIK3R1 mRNA levels in normal ovarian tissue and OC. p = 0.006 (One-way Anova).

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
6.
Figure 8

Figure 8. Immunostaining and gene copy number analysis of PTEN in OC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

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.

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
7.
Figure 5

Figure 5. FISH analysis of AKT1, AKT2 and PIK3CA genes in OC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Dual-colour fluorescence in situ hybridization analysis of AKT1 gene copy number. AKT1 gene, red signals; chromosome 14 centromere, green signals. Left, OC with cells polyploidy for chromosome 14; right, OC with amplification of the AKT1 locus. Original magnification 100X. B. Dual-colour fluorescence in situ hybridization analysis of AKT2 gene copy number. AKT2 gene, red signals; chromosome region 19p13.1, green signals. Left, OC with cells polyploidy for chromosome 19; right, OC with amplification of the AKT2 locus. Original magnification 100X. C. Dual-colour fluorescence in situ hybridization analysis of PIK3CA gene copy number. PIK3CA gene, red signals; chromosome region 3p14.1, green signals. Left, OC with cells polyploidy for chromosome 3; right, OC with amplification of the PIK3CA locus. Original magnification 100X.

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
8.
Figure 10

Figure 10. Analysis of pathways activated downstream PI3K in OC: HMGA1, JUN-B, FOS, MYC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Q-RT-PCR analysis of OCs with low or high expression of PIK3CA. B. Q-RT-PCR analysis of HMGA1 (top) and JUN-B (bottom) in OVCA429 (left) and TOV112D (right) treated with LY294002 (LY, 20 µM), RAD001 (RAD, 20nM) or a combination thereof for 24h. Values of mRNA are expressed as relative values using as standard the value of normal ovarian epithelial cells (IOSE 398).

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
9.
Figure 9

Figure 9. Analysis of pathways activated downstream PI3K in OC: mTOR and SGK3.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Immunostaining analysis of phosphorylated AKT, mTOR, S6K1, S6, and 4EBP1 in two representative formalin-fixed samples: left, p110α low (S17) and right, p110α high (S46). B. Protein from fresh-frozen samples were assayed by Western blot with the indicated antibodies. Samples S8, S17, S47, E2: low p110α expression; samples S63, E13: PIK3CA mutation; samples S63, S46, E3: p110α over-expression. Actin expression was used as a control for protein quality and loading. C. Representative pSGK3 immunostaining of pSGK3-positive (upper left panel) and pSGK3-negative (lower left panel) samples. Middle panels, immunostaining analysis of pAKT; right panels, immunostaining analysis of p110α of the same samples. Magnification 40X. Magnification of the insets 10X.

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.
10.
Figure 7

Figure 7. Mutation analysis of PIK3CA and KRAS genes in OC.. From: Multiple Genetic Alterations within the PI3K Pathway Are Responsible for AKT Activation in Patients with Ovarian Carcinoma.

A. Detection of mutations in PIK3CA by LightCycler (left) and direct sequencing (right). On the left, the negative derivative of the fluorescence (−dF/dT) versus temperature graph shows peaks with different Tm. The wild type sample showed a single Tm at 66°C. The heterozygous mutant sample showed an additional peak at 57°C. On the right, GAG→GCG transition in codon 545 of exon 9 inducing the substitution of a glutammic acid with an alanine (E545A). B. pAKT staining of a mutated S-OC (left) and E-OC (right). C. Point mutations in exon 2 of KRAS gene: GGT→GTT (G12V), GGT→CGT (G12R), GGT→ GAC (G13V). D. pAKT staining of a KRAS mutated sample (MU-6).

Carmela De Marco, et al. PLoS One. 2013;8(2):e55362.

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