MMP-9 activity not only with M-sh treatment in 4910 cells but also with other treatments. In addition, fibrin zymography revealed prominent reductions in uPA activity with all the treatments in 4910 cells (Fig. 1A) and further supported the fact that activity/expression of one protease influence the activity/ expression of other proteases and related molecules. Western blot analysis results clearly demonstrated significant reductions in protein expressions of these molecules with all the treatments except SV-sh compared to control cells (Fig. 1A–B). RT-PCR analysis of the cDNAs obtained from control and transfected 4910 cells revealed similar kind of reductions in these molecules at mRNA level (Fig. 1A). Taken together, these experiments at activity, protein and mRNA levels clearly demonstrate the efficiency of the shRNA plasmid constructs and the appropriate transfection conditions in xenograft cells. Reduction in the mRNA levels of MMP-9, uPAR and cathepsin B in M-sh, U-sh and C-sh transfected 4910 cells indicated a downregulation of MMP-9, uPAR and cathespin B target mRNAs, the mechanism by which shRNAs are proposed to work [24,25]. Bands related to GAPDH at mRNA and protein levels associated with various treatments in 4910 cells indicate that similar amounts of mRNA/protein were loaded in all treatment groups. Downregulation of proteases and uPAR inhibits adhesion to ECM proteins Cell adhesion to ECM is a crucial regulator of many functions, including cell cycle, survival, migration, proliferation, differentia- tion and ultimately, tumor invasion [4,26]. All the treatments (M-sh, U-sh, C-sh, MU-sh and MC-sh) inhibited the adhesion of both the xenografts to collagen, vitronectin, fibronectin and laminin. M-sh, U-sh and C-sh treatments in these xenografts significantly inhibited their adhesion to collagen (M-sh-39%, U-sh- 51% and C-sh-57% inhibition in 4910 cells and M-sh-40%, U-sh- 44% and C-sh-59% inhibition in 5310 cells), vitronectin (M-sh- 51%, U-sh-45% and C-sh-57% inhibition in 4910 cells and M-sh- 56%, U-sh-58% and C-sh-58% inhibition in 5310 cells), fibronectin (M-sh-36%, U-sh-48% and C-sh-48% inhibition in 4910 cells and M-sh-30%, U-sh-37% and C-sh-41% inhibition in 5310 cells), and laminin (M-sh-50%, U-sh-34% and C-sh-45% inhibition in 4910 cells and M-sh-48%, U-sh-52% and C-sh-65% inhibition in 5310 cells) (Fig. 1C). As expected, downregulation of MMP-9 in combination either with uPAR or cathepsin B further inhibited the adhesion of 4910 and 5310 cells to collagen, vitronectin, fibronectin and laminin (Fig. 1C). Results of the adhesion assay suggest bicistronic constructs that can simulta- neously downregulate two molecules are more effective in reducing the adhesion of xenografts to various ECM proteins when compared to the single constructs that can only downreg- ulate one molecule at a time. Interestingly, MC-sh treatment was found to be more effective than MU-sh in reducing the adhesion of both the xenografts to collagen and vitronectin. Integrins are transmembrane receptors for ECM proteins such as collagen, fibronectin, vitronectin and laminin. The classic role of integrins is to anchor cells to the ECM. Upregulation of a2b1, a3b1, a5b1, a6b1, aVb3 and a9b1 integrins in glioblastoma was previously reported [27–29]. Hence, in the present study, we assessed the effect of downregulating uPAR, MMP-9 and cathepsin B alone and in combination on integrin expression. Although we have performed RT-PCR analysis by using several alpha and beta integrin primers, here we presented/discussed only those integrins that are affected in 4910 cells with shRNA treatments. All the affected integrins in 4910 cells were tested in 5310 cells and subjected to all the shRNA treatments. RT-PCR analysis of b1 and b3 integrins in 4910 and 5310 xenografts Silencing Proteases in Glioma treated with various shRNAs for MMP-9, uPAR and cathepsin B revealed a marked reduction in their respective mRNA expression levels as compared to the controls (Fig. 2A). All the treatments downregulated the mRNA levels of b1 and b3 in both the xenografts except b1 and b3 mRNA levels in M-sh-treated 4910 xenograft cells. However, a significant reduction in these b integrin mRNA levels was noticed in both the xenografts treated with bicistronic constructs that simultaneously downregulate MMP-9 and either uPAR or cathespin B (Fig. 2A). The mRNA levels of several alpha integrins were reduced with MMP-9, uPAR and cathepsin B downregulation (Fig. 2B). a6, a9 and aV mRNA levels were downregulated in both the xenograft cell lines under all the treatment conditions. Immunocytochemistry of b1 and b3 integrins in 4910 and 5310 xenografts subjected to various shRNA treatments revealed a marked reduction in their protein expressions with all the treatments (Fig. 3A). Transfection with a scrambled vector in either of the xenografts did not affect the expression of these integrins. Reductions in the protein expressions similar to mRNA levels were noticed with a6, a9 and aV integrins when the transfected cells were subjected to immunocytochemistry (Fig. 3B). a1, a2, a7 and a10 integrins were downregulated only in 4910 xenograft cells treated with U-sh, C-sh, MU-sh and MC-sh (data not shown). These findings were further strengthened by the immunoblot analysis results wherein aV, a6, a9, b1 and b3 integrin protein expressions were significantly reduced after MU- sh and MC-sh treatments in 4910 cells (Fig. 4A–B). Based on these results and the earlier reported data [27–29], we hypothesize the involvement of aVb3, a6b1 and a9b1 heterodimer combinations on the effects mediated by our shRNA treatments in the glioma xenografts studied in this present investigation. FACS analysis results clearly demonstrated that 4910 cells express a6b1, a9b1 and aVb3 integrin heterodimers (Fig. 4C). Immunohistochemical analysis performed on the slide tissue microarrays of clinical GBM samples revealed the presence of aVb3, a6b1 and a9b1 integrins on several GBM clinical samples compared to normal cerebrum (Fig. 4D). shRNA treatment inhibited the migrating and invasive potential of glioma xenografts In control and SV-sh-treated xenografts, 80–90% of the wound repair occurred within 16 h after creating the wound in 5310 and 4910 cells. Hence, the percent wound repair after treatments with M-sh, U-sh, C-sh, MU-sh and MC-sh was assessed at 16 h time point in 5310 and 4910 cells (Fig. 5A). We noticed significant reduction in percent wound repair with all the treatments in both the xenografts. Quantification of the results revealed that the wound reduced to 38%, 57%, 54%, 29% and 1% in 5310 and 51%, 54%, 45%, 43% and 32% in 4910 cells treated with M-sh, U-sh, C-sh, MU-sh and MC-sh, respectively (Fig. 5C). As assessed by spheroid migration assay, downregulating individual molecules using M-sh, U-sh and C-sh reduced migration of cells from the spheroids (Fig. 5B). Significant and prominent reductions in migration of the cells from spheroids were noticed upon treatment with bicistronic constructs MU-sh and MC-sh (Fig. 5D). Quan- tification of migration of the cells from spheroids revealed 67%, 83%, 58%, 61% and 23% reduction in 4910 and 50%, 53%, 50%, 38% and 30% reduction in 5310 cells treated with M-sh, U-sh, C- sh, MU-sh and MC-sh, respectively (Fig. 5D). The most dangerous feature of malignancy is invasive and metastatic potential. Simultaneous downregulation of MMP-9 along with either uPAR or cathepsin B reduced the invasive potential of 4910 and 5310 cells through the Matrigel (Fig. 5E). Quantification of Matrigel invasion assay results revealed a significant reduction in invasive PLoS ONE | www.plosone.org 3 July 2010 | Volume 5 | Issue 7 | e11583