Silencing Proteases in Glioma Figure 3. Effect of shRNA plasmid constructs on the expression of integrins at protein level. (A) Immunocytochemistry was performed to evaluate the effect of various shRNA treatments on the expressions of b1 and b3 integrins. Microscopic images demonstrate b1 and b3 integrin expressions (red fluorescence) in 4910 and 5310 cells transfected with scrambled vector (SV-sh), MMP-9 (M-sh), uPAR (U-sh), cathepsin B (C-sh), MMP- 9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh). All treatments reduced b1 and b3 integrins expression. (B) The effect of various shRNA treatments on the expressions of a6, a9 and aV integrins was also evaluated and the microscopic images demonstrate a6, a9 and aV integrins expression in 4910 and 5310 cells transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh and MC-sh. All the treatments reduced a6, a9 and aV integrins expression in both the cell lines. doi:10.1371/journal.pone.0011583.g003 integrins involvement, we sought to determine the effect of these shRNA treatments on the signaling molecules associated with integrin mediated migration. Hence, we evaluated the effect of MU-sh and MC-sh treatments on several molecules such as FAK, talin1, talin2, profilin, vinculin, actinin, tensin, Rac1 and Arp2 in both the xenograft cell lines by using RT-PCR analysis. Downregulation of FAK, talin1, talin 2, profilin, tensin and Arp2 was noticed in treated 5310 cells whereas in 4910 cells, FAK, talin1, talin2, vinculin, actinin, tensin and Arp2 levels were reduced with both MU-sh and MC-sh treatments (Fig. 8A). Rac1 mRNA levels reduced only in MC-sh treated 5310 cells. Further, vinculin and actinin mRNA levels reduced only in 4910 cells treated with these bicistronic constructs. The expressions of FAK, phospho-FAK, cSrc, phospho-Src, Arp2, F-actin, paxillin, phos- pho-paxillin at protein level were reduced with both MU-sh and MC-sh treatments in 5310 cells (Fig. 8B). Rac1, talin1, a-actinin and vinculin protein expressions were reduced only in MC-sh treated 5310 xenografts. MU-sh and MC-sh treatments did not affect cofilin levels. In agreement with our adhesion, migration and invasion assay results, a more pronounced downregulation was noticed in majority of the migration-related molecules with MC-sh as compared to MU-sh. Discussion Malignant gliomas are highly invasive neoplasms. They may make a permissive environment for invasion by synthesizing and depositing an autologus matrix that facilitates their own motility. The ECM, which is a key component of the tissue destroyed during tumor cell invasion, is a dynamic environment that has a PLoS ONE | www.plosone.org 5 July 2010 | Volume 5 | Issue 7 | e11583