Journal: PLoS ONE

Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

doi: 10.1371/journal.pone.0015864

Figure Lengend Snippet: Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression CDC2/cyclin B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.

Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

doi: 10.1371/journal.pone.0015864

Figure Lengend Snippet: (A) Cellular localization by immunofluoresence shows that 14-3-3 σ (Green) is located in the cytoplasm and CDC2 (Red) is present in the nucleus (Blue). Compared to O 2 sensitive A2780 cells, the level of 14-3-3 σ is higher and CDC2 is low in the O 2 insensitive HeyA8 cells. In the O 2 sensitive A2780, 14-3-3 σ is localized both in the nucleus and cytoplasm at 21% O 2 . (A dotted yellow line, outlines a representative nuclei to indicate relative localization of 14-3-3 σ and CDC2 in these cells). (B) Western blot analysis of nuclear and cytoplasmic fractions show low levels of 14-3-3 σ in the nucleus compared to cytoplasm, with increased amounts of 14-3-3 σ being present in the cytoplasm of the O 2 insensitive HeyA8 cells. The level of CDC2 is higher both in the nucleus and cytoplasm of the O 2 sensitive A2780, but present in lower amount only in the nucleus of O 2 insensitive HeyA8 cells. Histone H1 and β−actin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (C) Mitotic cells were determined by counting the cells that stained positively for a mitosis specific marker, Phospho-Histone H3 from the total cell population. Mitotic fractions present at 3% or 21% O 2 were counted in both A2780 and HeyA8 and represented as bar graph. A significant increase in mitotic index (p>0.001, indicated by asterisk) was observed in the O 2 sensitive A2780 at 3% O 2 , but not in the O 2 insensitive HeyA8 cells. (D) Over-expression of 14-3-3 σ in the O 2 sensitive A2780 (Western Blot) results in loss of O 2 sensitivity (Bar graph). For the cells transfected with empty vector (mock transfection) or 14-3-3 σ over-expression construct, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2 (ambient), and (E) in the converse experiment performed with O 2 insensitive HeyA8, reducing the levels of 14-3-3 σ by siRNA (Western blot) results in restoration of O 2 sensitivity (Bar graph). For the cells transfected with scrambled siRNA (mock transfection) or siRNA against 14-3-3 σ, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2.

Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

Techniques: Western Blot, Staining, Marker, Over Expression, Transfection, Plasmid Preparation, Construct

Journal: PLoS ONE

Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

doi: 10.1371/journal.pone.0015864

Figure Lengend Snippet: (A) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 57 ovarian cancer cell lines. (B) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 205 ovarian tumors. The color codes for overall survival represents overall survival >24 months (blue) and overall survival <24 months (pink). The color codes for tumor stage represent stage I (red), stage II (green), stage III (light-blue) and stage IV (dark-blue). (C). Kaplan-Meier survival curve for the RPPA results comparing the group of ovarian tumors with high Phospho-RB and high 14-3-3 σ or CDC2 (blue line) with other expression profiles (red line).

Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

Techniques: Expressing