067 and 0.587 ± 0.182, respectively (Fig. 1E). Difference between Group1 and Group2 or Group1 and Group3 was significant (n = 3, P < 0.05). There is no difference between Group2 and Group3 (n = 3, P > 0.05). Data of the above experiments showed that the highest metastatic potential MHCC-97H cells expressed lowest level of PDCD4. The expression

level of PDCD4 was inversely correlated with the metastasis potentials of HCC cells. Plasmid construction and efficiency selleckchem of PDCD4 transfection A plasmid pcDNA3.1 (-)-PDCD4 encoding the PDCD4 gene was constructed. The recombinant was identified by double digestion with restriction enzymes and sequencing analysis. DNA sequencing of the recombinant pcDNA3.1 (-)-PDCD4 was also identified by Sangon. The efficiency of PDCD4 gene transfection was identified by western

blot analysis (Fig. 2A). Figure 2 Effects of PDCD4 on Compound C datasheet MHCC-97H cell proliferation and apoptosis. A: Western blot analysis for identification of transfection efficiency. B: MTT assay for cell proliferation. C: Flow cytometric assay for cell apoptosis. D: Hoechst 33258 staining for cell apoptosis (×200). Morphological changes of cell apoptosis were shown as chromatin condensation and nuclear fragmentation. Representative images are shown from three individual experiments. In C and D, a or Group1, b or Group 2, and c or Group3 represents cells of MHCC-97H-PDCD4, MHCC-97H-vector and MHCC-97H, respectively; d shows statistical analysis for each assay. Bars represent the means ± SD. The difference between Group1 and Group2 or Group3 was significant (P < 0.01). Effects of PDCD4 on MHCC-97H cells proliferation The MHCC-97H cell proliferation rate was assayed by MTT. The detected absorbance at 490 nm of the MHCC-97H-PDCD4 group was 0.543 ± 0.150, which was lower than that of the MHCC-97H-vector group (1.343 ± 0.268) or MHCC-97H group (1.278 ± 0.258). The difference was significant (n = 3, P < 0.05). No statistical

difference was found between the two control groups (n = 3, P > 0.05) (Fig. 2B). To further testify the Small molecule library effect of PDCD4 on proliferation of HCC cells, cell cycle analysis with a flow cytometer was performed and the proliferative indexes (PI) were calculated. As shown in Table 1, an increase of percentage both in G1 stage and in G2 stage was observed in MHCC-97H-PDCD4 cells, accompanied by a corresponding reduction in Montelukast Sodium the percentage of cells in S phase. PI was 27.83 ± 0.95%, 42.47 ± 2.90% and 44.47 ± 2.37% for the MHCC-97H-PDCD4 cells, the MHCC-97H-vector and the MHCC-97H cells, respectively. The difference of G1, G2, or S percentage and PI between the MHCC-97H-PDCD4 cells and the MHCC-97H-vector or the MHCC-97H cells is significant (n = 3, P < 0.05). No significant difference was found between the MHCC-97H-vector and the MHCC-97H cells. These data indicate that PDCD4 might promote both G1 and G2 arrest in MHCC-97H cells and further block the proliferation of HCC cells.