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. 2011 Jan 10;6(1):e15864. doi: 10.1371/journal.pone.0015864

Figure 5. 14-3-3 σand O2 sensitivity.

Figure 5

(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 O2 sensitive A2780 cells, the level of 14-3-3 σ is higher and CDC2 is low in the O2 insensitive HeyA8 cells. In the O2 sensitive A2780, 14-3-3 σ is localized both in the nucleus and cytoplasm at 21% O2. (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 O2 insensitive HeyA8 cells. The level of CDC2 is higher both in the nucleus and cytoplasm of the O2 sensitive A2780, but present in lower amount only in the nucleus of O2 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% O2 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 O2 sensitive A2780 at 3% O2, but not in the O2 insensitive HeyA8 cells. (D) Over-expression of 14-3-3 σ in the O2 sensitive A2780 (Western Blot) results in loss of O2 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% O2 (ambient), and (E) in the converse experiment performed with O2 insensitive HeyA8, reducing the levels of 14-3-3 σ by siRNA (Western blot) results in restoration of O2 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% O2.