Oxidative stress is elevated in tumors grown in Fbln5−/− mice.

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Oxidative stress is elevated in tumors grown in Fbln5−/− mice. Oxidative stress is elevated in tumors grown inFbln5−/−mice. (A) Fresh frozen sections of subcutaneous Pan02 tumors from WT (n=3) and Fbln5−/− (n=3) animals were incubated with dihydroethidium (DHE) for in situ detection of ROS. The resulting DHE-mediated fluorescence was quantified by Metamorph software. A representative image from tumors grown in WT and Fbln5−/− animals is shown. The results shown are the mean fluorescence intensity (±s.e.m.) from at least five 200×fields from each tumor. (B,C) The presence of DNA damage in frozen sections of subcutaneous Pan02 tumors from WT (n=5) and Fbln5−/− (n=7) animals was detected by immunohistochemistry with an antibody specific for 8-OHdG (B) and γH2AX (C) followed by development with a fluorescently conjugated secondary antibody. A representative image from tumors grown in WT and Fbln5−/− animals is shown. The results shown are the mean fluorescence intensity (±s.e.m.) from at least five 400×fields from each tumor stained. (D) Pan02 tumors from WT (n=3) and Fbln5−/− (n=3) mice were sectioned and stained with MECA-32 (green) for blood vessel identification and co-localized with anti-cleaved caspase 3 antibody (red) to determine apoptotic endothelial cells. The total number of dying endothelial cells was determined by hand counting and graphed as a percentage of the total number of apoptotic cells. Arrowheads indicate double positive cells. Ten pictures were taken from each tumor section (total magnification, 200×) for analysis. Results shown are ±s.e.m. *P<0.05, **P<0.01. Bars, 50 μm. Marie K. Schluterman et al. Dis. Model. Mech. 2010;3:333-342 ©2010 by The Company of Biologists Limited