Effect of SSHE on cell cycle and viability of pancreatic cancer cell lines.

<p>(A) Cell cycle. Panc-1 cells were treated with vehicle alone, 100 μg/ml SSHE or 200 μg/ml SSHE for 20 h, fixed, stained with PI, and then subjected to cytometry. (B) Cell viability. Pancreatic cancer cell lines were treated with vehicle alone or various concentrations of SSHE for 42 h. Cell viability was assessed by the MTT assay. Bars; SD. (C) Effect of SSHE on normal cells. HUVEC, HPAEpiC and Panc-1 cells were treated with vehicle alone or 100 μg/ml SSHE for 38 h. Cell viability was assessed by the MTT assay. Bars; SD. (D) Morphology. Panc-1 cells were treated with vehicle alone (left) or 200 μg/ml SSHE for 40 h (right). (E) Effect of SSHE on Panc-1 cells under hypoxic conditions. Panc-1 cells were treated with vehicle alone or various concentrations of SSHE for 42 h. Cell viability was determined by the MTT assay.</p

ROS production in SSHE-treated Panc-1 cells.

<p>(A) Panc-1 cells treated with vehicle alone, 100 μg/ml SSHE or 200 μg/ml SSHE for 20 h were stained with 10 μM H2DCFDA for 10 min and immediately observed under a confocal laser microscope. (B) Panc-1 cells treated as in A were subjected to cytometry. (C) Effect of NAC on SSHE-induced cell death. Panc-1 cells were treated with 200 μg/ml SSHE in the presence or absence of 10 mM NAC for 42 h. Cell viability was assessed by a trypan blue dye exclusion test. Bars; SD.</p

Antitumor effect of SSHE in the orthotopic model of Panc-1 cells.

<p>(A) Experimental set-up. (B) Bioluminescence imaging on Day 35. Left, IVIS images. Right, Quantitation of photons. (C) Body weight. Control group; n = 6, SSHE group; n = 5.</p

Antitumor effect of SSHE in the peritoneal dissemination model of Panc02 cells.

<p>(A) Experimental set-up. (B) Bioluminescence imaging on Day 15. Left, IVIS images. Right, Quantitation of photons. (C) Kaplan-Meier survival curve. (D) Body weight.</p

Effect of SSHE on the features of autophagy in Panc-1 cells.

<p>(A) Effect of SSHE on the expression of autophagy-related proteins. Panc-1 cells were treated with vehicle alone, 100 μg/ml SSHE or 200 μg/ml SSHE for 24 h. The cell lysates were subjected to Western blot analysis with anti-SQSTM1/p62 or anti-LC3 antibody. β-Actin served as a loading control. (B) Time-course of the conversion of LC3-I to LC3-II. Panc-1 cells were treated with 200 μg/ml SSHE for the indicated times. (C) AMPK and mTOR expression. Panc-1 cells were treated with 200 μg/ml SSHE for various times. The cell lysates were subjected to Western blot analysis with anti-AMPK, anti-phospho-AMPK antibody, anti-mTOR, or anti-phospho-mTOR. β-Actin served as a loading control. (D) AMPK activation and mTOR inhibition by SSHE. The intensity of the bands in (C) was quantified by Image J software. (E) Effect of 3-methyladenine (3-MA) and chloroquine (CQ) on SSHE-induced cell death. Panc-1 cells were treated with SSHE at the indicated concentration in the absence or presence of 10 μM 3-MA (left) or 40 μM CQ for 42 h. Cell viability was assessed by the MTT assay. Western blot images (A-C) have been cropped for presentation. Full size images are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126605#pone.0126605.s013" target="_blank">S13 Fig</a> Bars; SD.</p