Fig. 5. PP75-stathmin siRNA delivery in vivo.

Subcutaneous flank tumors were established by implantation of U251 cells into immunodeficient nude mice. On days 5 and 6 following the appearance of tumors, tumors were injected with PP75 alone (PP75), STMN siRNA alone (STMN siRNA), PP75-stathmin siRNA conjugates (PP75-STMN siRNA) or PP75-scrambled siRNA conjugates (PP75-scr siRNA). (A) Tumors were removed two days following treatment and relative stathmin mRNA expression levels were determined. The mean expression values of three tumors for each condition are shown. The error bars represent the standard deviation and the asterisk indicates p < 0.005 in comparison to PP75 treated tumors. (B) The tumors analyzed in fig. 5A were analyzed for stathmin protein expression by immunoblotting using an anti-stathmin antibody. Relative signal intensity of stathmin bands compared to β actin protein loading control bands are shown below the immunoblot image. Blots were performed in triplicate and representative results are shown. (C) The tumors analyzed in fig. 5A were also assessed for stathmin protein expression by immunohistochemistry using a primary rabbit anti-stathmin antibody and a fluorescently labeled anti-rabbit secondary antibody (red signal). Sections were counterstained with DAPI to reveal cell nuclei (blue). Representative sections from each treatment condition are shown and the scale bars correspond to 20 μm. (D) Two days following tumor treatment, animals not sacrificed for stathmin mRNA and protein expression analyses received intraperitoneal injection of the chemotherapeutic agent carmustine. Tumors were measured every 3 to 4 days and calculated mean volumes are shown with error bars representing standard deviations. PP75-STMN siRNA treated tumors significantly differ from each of the three control treated groups (*p < 0.001; two-way ANOVA).