The luminescent probe L-012 (Wako Chemical) was administered intravenously (40 mg/kg) after in vivo hypoxic treatments for in vivo ROS analysis. At 5 min after probe administration, luminescence from the animals was recorded with the IVIS Imaging System 200 Series (Caliper Life Sciences). To image HIF- 1 activity, mice were injected with 9.256106 Bq 18F-9-(4-fluoro-3- hydroxymethyl- butyl) guanine (FHBG) and imaged on a smallanimal PET scanner (microPET; Concorde Microsystems). In vivo GFP and DsRed expression were measured in an IVIS imaging system 200 series with excitation at 445-490 nm and emission at 515-575 nm for GFP or excitation at 500-550 nm and emission at 575-650 nm for DsRed. The image capture condition was set up as binning (868), f2, FOV13, 3 s. Signal intensity after background subtraction was quantified by Living Imaging software. For in vivo bioluminescence imaging (BLI) of tumor progression, mice were anesthetized with isoflurane and imaged 15 min after intraperitoneal injection of luciferin. Signal intensity was quantified within a region of interest over the head that was defined with LivingImage software. A perfusion marker, Hoechst 33342 (1 mg/mouse; Sigma), was intravenously (i.v.) administered 30 min prior to tumor excision. Tumor tissues were frozen in the OCT embedding matrix (Shandon Lipshaw). Frozen tissue sections (10 mm) were obtained with an OTF Publications Using Abomle AZ960 cryomicrotome (Bright-Hacker), fixed in ice-cold methanol for 10 min, and washed with PBS. Tumor sections were co-stained for Nox4 by including Nox4 antibody (Novus) at a final concentration of 10 mg/mL. Sections were washed 3 times in PBS, each wash lasting 5 min. For Nox4 staining, sections were incubated with DyLght 649-conjugated goat anti-rabbit antibody (1:100; Molecular Probes) and washed again. Tissue fluorescence was visualized with the Axio Observer A1 Publications Using Abomle Irinotecan digital fluorescence microscope system (ZEISS). Tumor tissues were disaggregated with an enzyme cocktail containing collagenase type III (Sigma), hyaluronidase (Sigma), and collagenase type IV (Sigma), washed several times, and resuspended in phosphate-buffered saline (PBS) to produce a single cell suspension.