Figure 8.

Targeting GBP1 inhibits glioblastoma invasion in vivo. (A) Western blot analysis of MMP1 and GBP1 in cell lysates (CL) and of MMP1 in conditioned medium (CM) of SNB19-shGFP and -shGBP1 cells with or without 20 ng/ml EGF stimulation for 24 h. (B) RT-qPCR analysis of GBP1 (left) and MMP1 (middle) mRNA expression in SNB19-shGFP and SNB19–shGBP1 cells with or without PBS or 20 ng/ml EGF stimulation for 6 h. (right) The human MMP1 promoter activity was determined by transfecting with pGL3-MMP1 (2,942 bp) and pRL-TK (internal control) in the shRNA-transfected SNB19 cells with or without PBS or 20 ng/ml EGF treatment for 6 h. Firefly and Renilla luciferase activities were measured, and promoter activity is presented as the fold induction of RLU as compared with the control. This result is expressed as the mean of three independent experiments ± SD. *, P < 0.05; #, P < 0.01. (C) H&E, GBP1, and MMP1 staining of brain sections on day 20 after intracranial inoculation of SNB19-shGFP (left) or SNB19-shGBP1 (right; 1 × 10⁶ cells/mouse). Shown are representative brain slices from tumor-bearing mice. Tumor margins are delineated using a dotted line. Arrowheads denote invasive extensions from tumor mass (T). Arrows indicate invasive tumor cells and disseminated tumor clusters away from the tumor mass. The animal experiments were performed two independent times with 10 mice per group with similar results. (D) Representative image showing perivascular infiltrations in SNB19-shGFP (left) but not SNB19-shGBP1 (right) tumor-bearing mice. Bars, 50 µm. (E) Quantification of the infiltrating tumor masses observed in SNB19-shGFP and SNB19-shGBP1 tumor-bearing mice on day 20 (n = 10; *, P < 0.05). Data are representative of two independent experiments. Error bars represent SD.