Figure 2.

Bevacizumab treatment of an orthotopic xenograft model of GBM induces autophagy. A&B, Sections of xenograft GBM tumor from the G39 and G59 PDX tumors treated with bevacizumab or placebo were reacted with rabbit anti-cleaved caspase-3 (0.2 µg/ml) (green) and mAb anti-Sox2 (2.5 µg/ml) (red) antibodies, followed by Alexa-488 and Alexa-594-conjugated secondary antibodies, DAPI nuclear stain and confocal microscopy. Representative photographs of tumor are shown (A). Quantitation of the number of double-labeled cleaved caspase-3 and Sox2⁺ cells in five representative fields/tumor (B). Data are graphed as Box and Whisker plots. C–E, Sections of xenograft GBM tumor from the G39, G59 and G44 PDX tumors treated with bevacizumab or placebo were reacted with anti-LC3 (2ug/ml) and anti-LAMP2 (4ug/ml) antibodies, followed by Alexa-488 and Alexa-594-conjugated secondary antibodies, DAPI nuclear stain and confocal microscopy. Representative photographs of tumor are shown (C). Quantitation of the number of LC3-LAMP2-positive puncta/cell in 10 representative fields/tumor (D); the adjusted means are 2.1 versus 0.8 for bevacizumab-treated versus placebo-treated tumors, respectively (p=0.02). Quantitation of the area of LC3-LAMP2-positive puncta/cell in10 representative fields/tumor (E); the adjusted means are 0.35 versus 0.12 for bevacizumab-treated versus placebo-treated tumors, respectively (p=0.02). Data are graphed as Box and Whisker plots; the green dots represent the adjusted means. In panel D, two data points in the bevacizumab-treated group (10.36 and 14.73) were removed for graphing purposes, but were included in the statistical analysis. In panel E, one data point in the bevacizumab-treated group (3.23) was removed for graphing purposes, but was included in the statistical analysis. Statistical analyses panels B, D and E: linear mixed model.