Ls in comparison to the untreated cells, respectively (p < 0.05 and p
Ls in comparison to the untreated cells, respectively (p < 0.05 and p < 0.01, Figure 8). Similarly, both Cyclin D1, Bcl-2 and Survivin protein expressions were measured by using Western blot analyses (Figure 9A). The protein levels of Cyclin D1 and Bcl-2 were downregulated by 32.62 ?2.38 , 29.12 ?3.05 and 45.78 ?2.54 (Figure 9B); 36.34 ?3.05 , 38.13 ?2.19 and 47.92 ?1.15 (Figure 9C), and Survivin protein expressions were decreased by 42.70 ?2.56 , 43.05 ?3.84 and 56.92 ?2.05 (Figure 9D) in VEGFsiRNA#1, KSP-siRNA#2 and siRNA cocktail transfected Hep3B cells compared to the untreated cells, respectively (p < 0.05 and p < 0.01, Figure 9). siRNA cocktail showed greater decrease of Cyclin D1, Bcl-2, Survivin expression at both mRNA and protein levels in comparison to VEGFsiRNA#1 or KSP-siRNA#2 alone (p < 0.05 and p < 0.01,Doan et al. Biological Research 2014, 47:70 http://www.biolres.com/content/47/1/Page 6 ofFigure 4 Effects of different treatments on the growth and the colony formation in Hep3B cells. (A) The proliferation of Hep3B cells was measured using WST-1 kit. The growth curve of Hep3B cells was shown for each group. The proliferation was assayed in triplicates at 0, 24, 48 and 72 hour post-transfection of siRNAs. (B) Effects of different treatments on the inhibition of cell proliferation were confirmed by the total numbers of colony. (C) Representative images of the colony formation assay were shown. Values were given as mean value ?standard deviation (SD) of triplicate. **p < 0.01, *p < 0.05 compared to untreated cell group; #p < 0.05 compared to siRNA cocktail treated cell PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 group.Figures 8 and 9). There was no significant difference in mRNA and protein levels of Cyclin D1, Bcl-2 and Survivin between CONT-siRNA treated cells and untreated ones.Effects of VEGF-siRNA#1, KSP-siRNA#2 and siRNA cocktail on tube formation in HUVECsA HUVECs angiogenesis model was employed to evaluate the tube formation of HUVECs stimulated by the conditioned medium derived from Hep3B cells transfected with siRNA cocktail, VEGF-siRNA#1, KSP-siRNA#2 and CONT-siRNA. As illustrated in Figure 10, siRNA cocktail or VEGFsiRNA#1 transfected Hep3B cells inhibited HUVECs to form extensive and enclosed tube networks on EPZ004777 price Matrigel as compared to the CONT-siRNA treated cells and untreated ones (p < 0.05, Figure 10B). However, KSP-siRNA#2 treated cells did not affect on tube formation in HUVECs. We also determined the mRNA and protein levels of ANG2 in HUVECs. In normally cultured negative controlcells, the expression of ANG2 mRNA (11.24 ?2.15 ) and protein (18.24 ?1.88 ) was slight, when compared to the untreated cells (Figure 11). CONT-siRNA did not cause any statistical differences compared to untreated cells. In VEGF-siRNA#1 treated cells, the expression of ANG2 mRNA (41.66 ?3.03 , p < 0.05, Figure 11A) and protein (59.62 ?1.84 , p < 0.05, Figure 11B) was significantly reduced compared to untreated cells. siRNA cocktail treated cells (ANG2 mRNA: 39.82 ?2.78 ; protein: 53.86 ?1.84 ) exhibited similar effect with VEGF-siRNA#1 treated cells. In contrast, the result was not reproduced by KSPsiRNA#2, which showed no significant difference in ANG2 expression in HUVECs between KSP-siRNA#2 treated cells and untreated ones (Figure 11).Discussion As tumor cells are characterised by multiple genetic and epigenetic alterations, the single inhibition of one tumourassociated gene as a therapeutic strategy may not beDoan et al. Biological Research 2014, 47:70 http://www.bio.