Identification of apoptotic cells by AO/EB staining.

<p>HeLa cells were treated with different concentrations of EPSAH (50–800 µg/ml) for 48 h and compared with untreated cells. The most representative fields are shown.</p

Agarose gel etectrophoresis of DNA from HeLa cells treated for 48 hours with different concentrations of EPSAH.

<p>Agarose gel etectrophoresis of DNA from HeLa cells treated for 48 hours with different concentrations of EPSAH.</p

Morphological changes in HeLa cells treated with different concentrations of EPSAH (50–800 µg/ml) for 48 h and compared with untreated cells.

<p>The cells were examined under the light microscope after Wright-Giemsa stain. Nuclear fragmentation, chromatin condensation and/or chromatin margination was observed in EPSAH-treated HeLa cells. The most representative fields are shown.</p

Effect of EPSAH on the cell proliferation in human HeLa cells.

<p>HeLa cells were seeded in 96-well culture plates. After incubation for 24, 48 or 72 h, they were subjected to MTT assay. Results are expressed as percent cell proliferation relative to the proliferation of control. Data represents as mean ± S.D. from three independent experiments. *<i>P</i><0.01 <i>vs</i> control.</p

EPSAH induced Bcl-2, Bax, P53, survivin, Grp78 and CHOP protein expression levels in HeLa cells.

<p>Cells were treated with EPSAH at the concentrations of 100 and 400 µg/ml for 48 h. The treated and untreated cells were lysed and cell extract subjected to western blots with the antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087223#s2" target="_blank">Methods</a> section. Immunoblot images are the representative of 3 identical experiments. The GAPDH blot is a loading control. (A) Bax/Bcl-2 proteins, (B) P53 and survivin proteins (C) Grp78/CHOP proteins.</p

EPSAH-induced mitochondrial transmembrane potential in HeLa cells measured by Rh123 staining and analyzed by flow cytometry.

<p>EPSAH-induced mitochondrial transmembrane potential in HeLa cells measured by Rh123 staining and analyzed by flow cytometry.</p

Molecular Mechanisms of Exopolysaccharide from <i>Aphanothece halaphytica</i> (EPSAH) Induced Apoptosis in HeLa Cells

<div><p>The present study aims to investigate the pharmacological effect of the exopolysaccharides from <i>Aphanothece halophytica</i> GR02 (EPSAH) on the HeLa human cervical cancer cell line. HeLa cells were cultured in RPMI-1640-10% FBS medium containing with or without different concentrations of EPSAH. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was elevated with Wright-Giemsa staining, AO/EB double staining, and DNA fragmentation assay. Apoptosis-associated molecules from cultured HeLa cells were quantified using Western blot analysis. Our results suggest that EPASH induces apoptosis in HeLa cells by targeting a master unfolded protein response (UPR) regulator Grp78. Grp78 further promotes the expression of CHOP and downregulates expression of survivin, which leads to activate mitochondria-mediated downstream molecules and p53-survivin pathway, resulting in caspase-3 activation and causing apoptosis. These findings provide important clues for further evaluating the potential potency of EPSAH for use in cancer therapy.</p></div

A potential mechanism of EPSAH-induced cell apoptosis in HeLa cells.

<p>(↑) and (↓) arrows indicate increase and decrease.</p

Effect of EPSAH on the level of the cleaved caspase-3 in HeLa cells.

<p>The level of the cleaved caspase-3 were assayed by Western blotting in the cells treated with EPSAH at the indicated doses for 48 h.</p