Silencing Proteases in Glioma Figure 2. RT-PCR analysis of integrins. (A) RT-PCR analysis of 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) plasmid shRNAs, showing the mRNA expressions of b integrins (b1 and b3) (A) and a integrins (a1, a2, a6, a7, a9, a10, and aV) (B). doi:10.1371/journal.pone.0011583.g002 potential of xenografts when subjected to treatment with either of the bicistronic constructs (Fig. 5F). Involvement of a9b1 on the migration potential of xenograft cells Compared to aVb3 and a6b1 integrins, very little is known about the role a9b1 integrin in glioma. We studied the effect of a9b1 blockade on the migratory potential of 5310 cells that are transfected with uPAR, MMP-9 and cathepsin B expressing plasmid DNAs. RT-PCR analysis of uPAR, MMP-9 and cathepsin B in 4910 and 5310 cells transfected with uPAR expressing (U-fl), MMP- 9 expressing (M-fl) and cathepsin B expressing (C-fl) plasmid DNAs clearly demonstrated the efficiency of the constructs and transfec- tions (Fig. 6A–B). As expected, significant increase in percent wound repair noticed with U-fl, M-fl and C-fl treatments in 5310 cells which was significantly blocked with the same treatments in presence of a9b1 antibody (Fig. 6C–D). Furthermore, a9b1 integrin levels as revealed by FACS analysis were significantly reduced after MU-sh and MC-sh treatments in 4910 cells (Fig. 6E). These results clearly demonstrate the involvement of a9b1 in uPAR, MMP-9 and cathepsin B mediated migration in glioma xenograft cells. Effect of bicistronic constructs on proliferation in vitro and tumors in vivo MU-sh and MC-sh treatments efficiently reduced the colony forming ability of 4910 and 5310 cells (Fig. 6F). Similarly, proliferation of 4910 cells as evidenced by MTT assay was significantly reduced on Days 4, 5 and 6 after MU-sh and MC-sh treatments compared to untreated 4910 cells (Fig. 6G). Hematoxylin and eosin staining performed on mouse brains (injected with xenograft cells 2 weeks before treatment) revealed a prominent reduction of tumor growth in MU-sh and MC-sh treated mouse brains when compared to the controls (Fig. 7A). Aggressive tumor formation was noticed in xenografts injected mouse brains compared to normal brains (Fig. 7A–B). Further- more, the apoptotic index of tumor cells quantified by the number of positive cells for TUNEL staining showed a drastic and significant increase in the number of apoptotic cells in the tumor sections from mice that were treated with bicistronic constructs (data not shown). Brains of the animals that were sacrificed 2–3 weeks prior to the end of the treatment were used to evaluate the effect of MU-sh and MC-sh treatments on the expression of aVb3, a6b1 and a9b1 integrins. As expected, prominent reduction of aVb3, a6b1 and a9b1 integrin expression was noticed on the 5310 in vivo tumors that were treated with MU-sh and MC-sh (Fig. 7C). Immunohistochemical analysis of platelet endothelial cell adhesion molecule (PECAM-1) performed on the brain sections of 4910 and 5310 control tumors and MU-sh and MC- sh treated 5310 tumors (from the animals that are sacrificed 2–3 weeks prior to the end of the treatment) revealed that the extent of blood vessels in 4910 and 5310 untreated tumors are more compared to MU-sh and MC-sh treated 5310 tumors (Fig. 7D). Effect of silencing proteases and uPAR on FAK and other migration molecules Our results clearly demonstrated the involvement of aVb3, a6b1 and a9b1 and integrins in the effects mediated by shRNA treatments and thereby affected the adhesive, migrating and invasive potential of glioma xenografts. To further confirm the PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11583