Stable CAL-101 nmr transfection is described in the Supporting Materials and Methods. Whole cell protein was extracted with cell lysis buffer (Sigma-Aldrich, St. Louis, MO). Cytoplasmic protein extraction and western blotting analysis were performed by following

a standard protocol, as described previously.17 The RNA interference experiment protocol is described in the Supporting Materials and Methods. HMGB1 level in serums from humans and mice was detected by enzyme-linked immunosorbent assay (ELISA) (IBL, Toronto, Ontario, Canada), according to the manufacturer’s instructions. Cultures were fixed, stained, and examined under a confocal microscope (Olympus, Tokyo, Japan), as described in the Supporting Materials and Methods. The Caspase-1 Colorimetric Assay kit (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s protocol. We determined migration and invasion as previously described.18 To examine the metastatic potential of stable HMGB1 knockdown

clones, 2 × 106 Hepa1-6, in 0.3 mL of phosphate-buffered this website saline, were injected into the tail vein of C57BL/6 mice. For in vivo tracking, the Hepa1-6 cells were stably transfected with firefly luciferase. One hundred milligrams per kilogram of D-luciferin (Caliper Life Sciences, Hopkinton, MA) were injected into the peritoneal cavities of mice, and bioluminescence was detected with the IVIS 100 Imaging System (Caliper Life Sciences). Results are expressed as the mean ± standard error of the mean (SEM). Statistical Phospholipase D1 analysis was performed using

the Student’s t test or one-way analysis of variance test. All statistical analyses were performed using Sigma Stat v.3.5 (Systat Software, Inc., Chicago, IL). Graphs were generated using Sigma Plot v.10 (Systat Software). P < 0.05 was denoted as statistically significant. Overexpression of HMGB1 is associated with tumor progression.13 To study the role of HMGB1 in HCC, we first examined the amount of HMGB1 in 20 HCC tissue samples and their corresponding nontumor liver by immunoblotting analysis. The detailed clinicopathological information of 20 cases is shown in Supporting Table 1. We found that the expression of HMGB1 was higher in all HCC tissues (Fig. 1A). Compared to normal primary hepatocytes, the expression of HMGB1 was also much stronger in five HCC cell lines (Fig. 1B). We then examined the level of nuclear and cytoplasmic HMGB1 by the fractionation of nuclear and cytoplasmic proteins in HCC tissues and nontumor liver tissues. The amount of nuclear HMGB1 in HCC tissues and nontumor tissues was not significantly different (data not shown). However, cytoplasmic HMGB1 was absent or present at low levels in nontumor tissues, whereas cytoplasmic HMGB1 was found at high levels in HCC tissues (Fig. 1C). High cytoplasmic levels of HMGB1 usually occur in the context of active HMGB1 release.