Cytoimmunochemistry and Immunohistochemistry 2×105 MHCC97-H and MHCC97-L cell were plated and cultured in six-well plate respectively, when reached to 60% confluent, the cells were fixed with 100% methanol, permeabilized with 0.5% Triton X-100, and sequentially incubated with the primary anti- TGF β1 monoclonal antibodies and anti-mouse

check details immunoglobulin (Ig) coupled to Horseradish peroxidase (HRP), then, the cells were stained with DAB (3, 3′-diaminobenzidine) and counterstained with hematoxylin. Paraffin-embedded tumor tissues were sliced as 5μm sections in thickness and mounted on glass. Slides were deparaffinated and rehydrated over 10 min through a graded alcohol series to deionized water; 1% Antigen Unmasking Solution (Vector Laboratories) and microwaved were used to enhance antigen retrieval; the slide were incubated with anti-TGF β1 monoclonal antibodies and HRP-conjugated secondary antibody, and then, stained with DAB. ELASA Total protein of all tumor tissues Apoptosis inhibitor were extracted as described above. TGF β1 protein levels in tumors were determined using the Quantikine TGF β1 Immunoassay (R&D, Minneapolis, MN,USA). The operational approach was performed according to manufacture specification. Statistical analysis Statistical analysis was performed using SPSS 11.5 software (SPSS Inc, USA). The data were analyzed by Students’ t test, one-way analysis of variance and covariance analysis. All statistical

tests were two-sided; a P value of less than Crenolanib in vitro Paclitaxel datasheet 0.05 was considered statistically

significant. Results The tumor weight and pulmonary metastatic rate The tumors of MHCC9-H model grew fast than that of MHCC97-L, and especially in early stage of tumor formation, MHCC9-H spent shorter time (days) than MHCC97-L getting to the size of 500mm3 (21.93±3.67 vs. 30.83±1.94, P<0.001) (Figure 1A), however, the growth speed became similar from the size of 500mm3 to 1500 mm3 (9.00±2.69 vs.10.83±1.47, P=0.14 ) (Figure 1B). MHCC9-H model had bigger pulmonary metastatic loci than MHCC97-L model (Figure 1C,D). The mean tumor weight (g) in MHCC9-H and MHCC97-L were 1.75±0.75 and 1.26±0.51, and the pulmonary metastatic rate were 55% and 36.36%; and the average number of metastatic cell in lung were 119.25±177.39 and 43.36±47.80 respectively (Table 1). Figure 1 Comparison of Growth and pulmonary metastsis in mice models. A) Growth curve of MHCC97-H and MHCC97-L models; B) Average days which were spent for getting to tumor size. * denoted P<0.05, Error bar represent the standard errors of the mean. C,D) MHCC97-L models (C) had smaller pulmonary metastatic loci than MHCC97-H models (D). Arrows denote metastatic loci. Table 1 The tumor weight and pulmonary metastasis rate in different nude mice models of HCC Models No. of cases Tumor weight(g) (Mean±SD) Metastatic rate No. of Metastatic cells (Mean±SD) MHCC97-L 11 1.26±0.51 36.36% (4/11) 46.36±47.80 MHCC97-H 20 1.75±0.75 55.00% (11/20) 119.25±177.39 SD=standard deviation.