Promising Noninvasive Cellular Phenotype in Prostate Cancer Cells Knockdown of Matrix Metalloproteinase 9

Figure 6

Analysis of the effects of CD44 knockdown on the expression of CD44v6, cell morphology, and actin distribution. (a), (b), (g), and (h) The effect of ShRNA to CD44 on the cellular levels of CD44s protein was determined by immunoblotting analysis. The expression levels of CD44s are shown in different PC3 cell lines by immunoblotting analysis (a). Individual clones were isolated from cells transfected with a construct corresponding to 492 bp ((a), lane 1) and 801 bp (lane 2). Cells transfected with a nonsilencing scrambled ShRNA construct (Sc; lane 3) and vector DNA (V; lane 4) were used as controls. PC3 cells knockdown of CD44s ((g), lane 1) and MMP9 (lane 2) as well as a control PC3/Sc cells (lane 3) were used for immunoblotting analysis with an antibody to CD44v6. Total cellular lysates (~50 μg) were used for immunoblotting analyses (a), (b), (g), and (h). Equal loading of protein was verified by immunoblotting with an antibody GAPDH (b) and (h). The experiment was repeated three times with similar results. (c)–(f) The morphology of PC3/Sc and PC3/Si (CD44) cells was determined by phase contrast microscopy (400x; (c) and (e)). Cells were stained with rhodamine phalloidin to examine the distribution of actin in PC3/Sc (d) and PC3/Si (CD44) (f) cells. Staining of actin is shown in gray scale (d) and (f). Arrows point to invadopodia in PC3/Sc cells (d). The results shown are representative of three independent experiments. Bar: 50 μm.