As revealed by AnnexinV-7/AAD double staining, when exposed to 45˚C for 10 minutes, more than 96% of HEPG2 and 91.6% HuH7 cells had survived after 24 hours without signs of apoptosis, whereas survival was decreased to 65.6%

and 87.6%, respectively, at 50˚C, and all cells died after exposure this website to 55˚C (Fig. 1A). Division indices (DI) of three HCC cell lines were determined after CFSE labeling, followed by FACS analysis. In all cell lines that were exposed to 50˚C, DI at day 5 (or 6) was significantly higher than in cells kept at 37˚C (Fig. 1B). This was paralleled by a significant increase of proliferation-related transcripts (Ki-67 and CyclinD1), with Ki-67 transcripts being already elevated in two cell lines after exposure to 45˚C (Fig. 1C). Distinct morphological changes, such as appearance of spindle-like cells (Fig. 2A), were only observed for HEPG2 cells exposed to 50˚C on day 5. Intracellular staining, followed by FCM, demonstrated that the cholangiocyte markers, CK7 and CK19, were

increased in HEPG2 cells at day 5 after exposure to 50˚C, whereas low baseline expression was observed in cells exposed to 37˚C or 45˚C (Fig. 2B). This was confirmed by western blotting for CK19 (Fig. 2C) and immunohistology (data not shown). Cell phenotype-related transcript levels were analyzed in the three HCC cell lines by using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR; Fig. 3). CK7 and 19 transcripts were dramatically elevated on day 5 day after exposure to 50˚C, compared to no or minor changes after treatment at lower temperatures, but this increase was transient Bortezomib and levels returned to or near baseline on day 12. Transcript levels of the putative stem cell and progenitor marker, CD133, showed similar kinetics, whereas the hepatocyte differentiation marker, albumin (ALB) was significantly reduced on day 5, to rise baseline on day 12. Expression of four central EMT markers (Snail, TWIST1, CHD1L, and COL1A1) was increased in all three HCC cell lines 5 days after treatment at 50˚C (Supporting see more Fig. 1). Most of these increases were significant or highly significant, especially for COL1A1. The transcript

level (cycle threshold [Ct] value) for COL1A1 almost resembled that found in the well-established, activated human hepatic stellate cell line, LX-2[33] (Supporting Fig. 2). Tissue inhibitor of matrix metalloproteinase, another EMT-related marker, showed a similar trend (Supporting Fig. 3). Of note, EMT-related transcript levels returned to baseline on day 12 post–heat treatment. EMT-like changes, enhanced invasiveness, and migration of HEPG2 cells were confirmed by a 5- to 8-fold increased protein expression of Snail at day 5, but also at day 12 after heat treatment, and a significantly enhanced level of in vitro HEPG2 and HuH7 invasions at day 5 (Fig. 4A,B). Preheating (50˚C) preheating increased Shc transcript levels 5.0-, 2.8-, and 5.1-fold at day 5 in HEPG2, HuH7, and HEP3B, respectively (Fig. 5A).