To assess whether following transfection of melanoma cells with the DDX11 siRNA, DDX11 mRNA would be downregulated, we transfected WM1158 MGP melan oma cells with 25 nM of the DDX11 siRNA not conju gated to Cy5, and serving as control, with add to your list 25 nM of a pool of siRNAs comprised of four non targeting siRNAs. Using a pair of qPCR primers spanning exons 20 22 of human DDX11, we performed qPCR analysis, which as depicted for the 48 hr time point post transfection, revealed that expression of DDX11 was decreased when compared with WM1158 MGP melanoma cells that had received only the siRNA delivery vehicle Lipofectamine 2000, or the pool of the control siRNAs. The most prominent phenotypic change we observed during this series of experiments was that compared with control siRNA transfected cells, melanoma cells that had been transfected with the DDX11 siRNA, exhibited a rapid and dramatic alter ation in their morphology.

Specifically, we found that as shown in Figure 2C, panels b d, transfection of the DDX11 siRNA caused melanoma cells to lose cell cell contact in as little as 24 hr, and at doses of 25 nM as well as 150 nM of DDX11 siRNA, a significant number of Inhibitors,Modulators,Libraries the transfected melanoma cells exhibited a tightly held together chain like morphology. Inhibition of DDX11 expression leads to chromosome segregation defects in melanoma cells To gain insights into the cause of the DDX11 siRNA induced morphologic changes, we transfected melanoma cells with the DDX11 siRNA and 24 hr later prepared chromosome spreads that were stained with either fluor escent DAPI or Giemsa solution.

Compared with the DAPI stained chromosome spreads of melanoma cells Inhibitors,Modulators,Libraries that had been transfected for 24 hr with the control siRNA pool, the DAPI stained chromosome spreads of DDX11 siRNA transfected melanoma cells Inhibitors,Modulators,Libraries revealed a pattern of tightly condensed chromosomes. This difference in chromosome abnormality became even more apparent when the chromosome spreads of control siRNA and DDX11 siRNA transfected mel anoma cells were stained with Giemsa. Since it has been reported that DDX11 plays an im portant role in maintaining fidelity of chromosome arm cohesion prior to mitosis, we next determined in a total of 40 chromosome spreads prepared from the DDX11 siRNA transfected and likewise, the control siRNA transfected melanoma cells, the number of chromosomes that had closed, partially closed, or open separated Inhibitors,Modulators,Libraries arms. As displayed in Figure 3C, panel a, compared with Inhibitors,Modulators,Libraries the control, the DDX11 siRNA transfected melanoma cells www.selleckchem.com/products/Y-27632.html exhibited approximately 50% fewer chromosomes with closed arms, and about 50 60% more chromosomes with partially closed or open separated arms.