All reagents and instruments were purchased from Applied Biosystems. The reaction master mix, containing 2× RT Buffer, 20× Enzyme Mix, and nuclease-free water, was briefly mixed with 20 ng of each total RNA sample. Mixtures were incubated for 60 minutes at 37°C, 5 minutes at 95°C, and then kept at 4°C. Real-time quantitative FDA approved Drug Library purchase reverse-transcription polymerase chain reaction (qRT-PCR) was carried out using the Applied Biosystems 7500 Real-Time PCR System. The PCR master mix, containing TaqMan 2× Universal PCR Master Mix, 20× TaqMan assay, and RT products in a 20-μL reaction volume, was processed as follows: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and then 60°C for 60 seconds. The

signal was collected at the endpoint of every

cycle. The mean values of the Ct, obtained Ibrutinib in vivo in duplicate or triplicate, were used for data analysis. Representative sections of formalin-fixed, paraffin-embedded tissues were used for IHC. Primary antibodies against K7, K19, EpCAM, CD56, alpha-fetoprotein (AFP), HepPar1, Smad4, and Snail were used (Supporting Table 1). We used the DAKO Envision Kit (Dako, Glostrup, Denmark) for IHC with a single primary antibody, then applied 3,3-diaminobenzidine (Dako). For double IHC staining, the EnVision AP system (Dako) and Vector Blue Alkaline Phosphatase Substrate Kit III (SK-5300; Vector Laboratories, Burlingame, CA) were used to detect the first primary antibody, then the EnVision DuoFLEX Doublestain System (SK110; Dako) and Vector NovaRED Substrate Kit (SK-4800; Vector Laboratories) were used to detect the second primary antibody. The expression of each marker was evaluated as positive when it was detected in more than 5% of tumor cells and was scored as follows: 1+ for detection in 5%-10% of tumor cells,

2+ for 11%-50%, and 3+ if detected in over 50% of tumor cells. Statistical analysis was performed using the SAS software (version 9.1.3; SAS Institute Inc., Cary, NC) and R package (http://www.r-project.org). We assessed the IHC stain results using the chi-square test, and the Student’s t test was used to compare the results of the real-time qRT-PCR. The bivariate correlation test MCE was used to analyze correlations among the qRT-PCR results. Survival analysis was carried out using Kaplan-Meier’s method, and differences were analyzed using the log-rank test. In histological evaluation, S-HCCs showed abundant fibrous stroma between trabeculae or solid nests of tumor cells, and CCs also showed marked fibrous stroma between tumor glands, whereas HCCs showed trabecular or adenoid patterns with no or little fibrous stroma (Fig. 1). The centers of the S-HCC nests were composed of polygonal cells with abundant cytoplasm resembling mature hepatocytes, whereas the periphery of the tumor nests facing the fibrous stroma was composed of small, oval-shaped tumor cells with a high nuclear cytoplasmic ratio (Fig. 1B).