2016;7:47431\47443. annealed by GenePharma (Shanghai, China). Two focus on sequences of CD44s siRNA, 518 and 688, were selected. These sequences were: 5\CCGCTTTGCAGGTGTATTC\3; and 5\AAATGGTCGCTACAGCATC\3, respectively. An siRNA with a non\targeting sequence (scrambled sequence) was used as a negative?control (shNC) in our ICG-001 experiment. RNA interference was carried out as described previously.11 2.9. In?vitro migration and invasion assays For in?vitro migration and invasion assays, cells were seeded onto the upper chamber of a transwell or ICG-001 on a Matrigel\coated transwell (BD Biosciences, Franklin Lakes, NJ, USA) in serum\free media. The lower chamber contained DMEM with 10% FBS as a chemoattractant. After 12 or 48?hours of incubation, non\migrated cells were gently removed from the upper chamber with a cotton swab. Cells were fixed and stained using Giemsa solution and counted in 5 randomly chosen visual fields. 2.10. Statistical analysis Statistical analyses were carried out using SPSS 16.0 software. All data are presented as the mean??SD. Two\group comparisons were analyzed using the two\tailed Student’s test. Three or more group comparisons were analyzed using one\way ANOVA. P?AKAP13 drugs in many tumor cell types. Therefore, we also investigated the effect of TM on HCC cell apoptosis. Increased apoptosis was observed in MHCC\97L cells treated with TM, implying that an increased rate of apoptosis could be one of the mechanisms of TM inhibition of cell proliferation (Figure?2A). To understand the mechanism by which TM induces cell apoptosis, we assessed the expression of Bcl\2 family proteins using western blotting. Results showed that the proapoptotic Bcl\2 family proteins?Bim and Bid were up\regulated, and that the concomitant anti\apoptosis proteins Bcl\xL and Mcl\1 were down\regulated in TM\treated ICG-001 MHCC\97L cells (Figure?2B). However, the expression of Bcl\2 and PDCD4 did not change after TM treatment. Open in a separate window Figure 2 Tunicamycin (TM) ICG-001 induces cell apoptosis and regulates the expression of Bcl\2 family proteins in hepatocellular carcinoma (HCC).

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