Bcr and Abr Regulate PH Figure 5. Loss of Bcr or Abr promotes hypoxia-induced Rac1 activation in vitro and in vivo. A, Real-time RT-PCR analysis for quantification of rac1, rac2 and rac3 mRNA in PASMCs. B, Represen- tative gel electrophoresis of RT-PCR products showing absence of rac2 in PASMCs. Samples loaded are indicated above the lanes; spleen, positive control; RNA (2), no RNA, negative control. C–F, Assay for Rac1 activation. C–D, Analysis of activation of Rac1 in vivo in the lungs of bcr2/2, abr2/2 and wt mice after normoxia or hypoxia exposure. E–F, Analysis of Rac1 activation in PASMC under normoxia or hypoxia. C and E, representative Western blots; D and F, quantification. Three independent samples of each genotype were tested and the entire experiment was repeated independently. To quantitate results, Western blots were scanned and the ratio of GTP-Rac/total Rac was determined (panels D, F). * p,0.05 when compared with the results of the same genotype under normoxia. # p,0.05 when compared with WT exposed to the same condition. doi:10.1371/journal.pone.0049756.g005 camera. The arterial areas were analyzed with ImageJ 1.41 software (NIH, Bethesda, MD) for percentage wall thickness. For immunohistochemistry, lung sections (5 mm) were depar- affinized. Vascular smooth muscle cells were detected by immunohistochemistry with a mouse monoclonal antibody to SMA, using a LAB-SA detection system (Invitrogen). Areas of cells positive for anti-SMA staining were quantified using ImageJ software (NIH). Briefly, contour plots were drawn around the a- SMA-stained cells. Images were then converted to grayscale format. A threshold was set for a-SMA-positive staining. Within the contour plots, pixels with intensities above the threshold were quantified and converted to areas using the scale bar on the image. Measurement of the Mean Linear Intercept For each image, 4 areas of interest, free of airways and blood vessels, were randomly picked for measuring the Mean Linear Intercept (MLI). A grid (565 with 20 mm between lines) was superimposed over the area of interest. The number of times (intercepts) that the alveoli intercepted the grid lines was counted. The MLI was calculated by multiplying the length of the lines and the number of lines per area, then dividing by the total number of intercepts [58]. The average alveolar area was quantified with Fiji ImageJ software [59]. The images were first processed with a median filter (radius 2.0 pixels), color deconvolution with an H&E 2 matrix, and an unsharp mask (radius 20 pixels, weight 0.9) on the H&E image. Default auto-thresholding and a binary fill-holes function were next applied. The area of all objects was measured with the 2 Analyze Particles function, excluding objects with areas ,60 mm . Objects representing trachea and blood vessels were manually excluded from the analysis. Data represent .100 measurements per genotype per condition. Measurement of ROS Production in Macrophages Mice were injected intraperitoneally with 4 ml of 4% thiogly- collate medium. 4 days after the injection, macrophages were harvested from peritoneal cavities. Macrophages in complete RPMI-1640 medium (10% FBS) were exposed to normoxia or hypoxia for 24 hours. Cells were then treated with PBS (control cells for basal ROS production) or activated with PMA (1 mM) for 1 hr. Measurement of ROS production was performed using a CellROX Deep Red Reagent kit (Life Technologies, CA) according to the manufacturer’s protocol. Data were collected using an Accuri flow cytometer (BD Biosciences). Evans Blue Vascular Leakage Assay Mice subjected to hypoxia for 3 weeks were injected with Evans blue (30 mg/kg body weight) via the tail vein. 20 min after the PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49756