and cultured at 37uC in 95% air/5% CO2. The PASMC phenotype was confirmed by morphological features and immu- nostaining for SMA. In vitro Cell Proliferation Assay and Flow Cytometric Cell Cycle Analysis PASMCs (3rd passage) from male bcr2/2, abr2/2 and wt FVB/J mice were seeded in 6-well plates (16104 cells/well) and cultured in serum-free medium for 24 hrs. Effective cell synchro- nization was confirmed by flow cytometry analysis. Triplicate wells of cells were then exposed to normoxia or hypoxia (5% O2), with or without 25 mmol/L of the Rac inhibitor Z62954982 (ZINC08010136; Enamine Ltd, Cincinnati, USA). Z62954982 was made as a concentrated stock solution in DMSO (dimethyl sulfoxide). The final concentration of DMSO in the culture medium was 0.1%; control wells were treated with 0.1% DMSO only. Z62954982 did not have cytotoxic effects, since viability of the drug-treated PASMCs was around 95%, comparable with that of PASMCs treated with 0.1% DMSO. Medium was refreshed every 2 days. After 5 days, the supernatants were collected for measurement of IL-6. Cells were trypsinized and counted. PASMCs were then fixed with 70% ethanol and incubated with staining solution (20 mg/ml propidium iodide, 0.2 mg/ml DNase- free RNase, 0.01% Triton-100 in PBS). Cell cycle was analyzed using a flow cytometer (Accuri Cytometers). The proliferation index of the PASMCs was calculated as: (S+G2/M)/(G0/G1+ S + G2/M) 6100%. G0/G1, S, G2/M represents the percentage of the cells in G0/G1, S, G2/M phase, respectively. Quantitative Real-time PCR For the isolation of RNA and protein, 3rd passage PASMCs from Bcr2/2, Abr2/2 and WT mice were plated in 10 cm culture dishes (36104/dish). Cells were treated either with DMSO or with Z62954982 (25 mmol/L, dissolved in DMSO, Enamine Ltd) in an incubator with normoxia or hypoxia (5% O2) for 5 days. Medium was refreshed every other day. Fresh Z62954982 was added along with the change of medium for the Z62954982 treatment cells. On day 5, cells were harvested, washed 3 times with PBS, harvested by scraping and processed for RNA (RNA mini kit, Qiagen) or for protein isolation in MLB (see below). 2 mg of RNA was converted to cDNA. The primer pairs used for amplification were: rac1, 5’- TGGCGAAAGAGATCGGTGCTGT-3’ (sense), 5’-TTCTTGACAGGAGGGGGACAGAGA-3’ (antisense); rac2, 5’-ACCTCCTAGCCACTCCATACCACT-3’ (sense), 5’-CACCACACACTTGATGGCCTGCAT-3’ (antisense); rac3, 5’-TGAGAATGTCCGTGCCAAGTGGT-3’ (sense), 5’-CCGCAGCCGTTCAATCGTATCCTT-3’ (antisense); Glyceraldehyde 3-phosphate dehydrogenase (gapdh), 5’-ACCCAGAAGACTGTGGATGG-3’ (sense), 5’-CACCACACACTTGATGGCCTGCAT-3’ (antisense). Amplification reactions were performed with SYBR Green (Invitrogen) in an ABI Prism 7700 thermal cycler sequence detection system (Perkin-Elmer, CA). To confirm specificity of amplification, the PCR products from each primer pair were subjected to a melting curve analysis and electrophoresis on 2% agarose gels. The relative mRNA levels of target genes to that of gapdh were calculated. Antibodies, Western Blotting Rac activation assays were carried out as described previously [7]. Briefly, right lobes of the lungs from normoxia or hypoxia treated mice were isolated, washed 3 times with TBS, and Bcr and Abr Regulate PH homogenized with Mg2+ lysis/wash buffer (MLB, 25 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Igepal CA-630, 10 mmol/L MgCl2, 1 mmol/L EDTA, 10% glycerol, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mmol/L sodium pervanadate, 1 mmol/L phenylmethylsulfonyl fluoride) on ice. These lysates were used for standard Western blotting and Rac activation assays. Lung lysates (in total, samples from 6 mice per genotype per condition) and PASMCs lysates in MLB (from triplicate wells and two indepen- dent experiments) were incubated with recombinant Pak1-GST PBD precoupled with glutathione-agarose beads at 4uC with rotation for 1 hour. Beads were washed 3 times with MLB and resuspended in SDS-sample buffer. Lysates to measure total Rac1 were collected before performing the Rac activation assay. Immunoblotting was performed with anti-Rac1 antibodies (1:1000, Cytoskeleton). Anti p38 and p-p38 antibodies (1:500) were from Cell Signaling. Abr antiserum (1:200) has been previously described. [8] Bcr (N-20; 1:500) antibodies were from Santa Cruz and GAPDH antibodies (1:5000) from Millipore. Anti- phosphotyrosine antibodies were obtained from BD Biosciences. Western blot results were quantified using Un-Scan-It software (Silk Scientific, Orem, UT) and analyzed as the ratio of phosphorylated p38 MAPK to total p38 MAPK or of GTP- bound Rac1 to total Rac. Western blot analysis was done with Abr antiserum (1:200) [8] and Bcr (N-20)(1:500, Santa Cruz) or GAPDH antibodies (1:5000, Millipore). ELISA The IL-6 levels in the serum and lungs of mice and PASMCs supernatants were measured by Enzyme-Linked Immunosorbent Assay (ELISA) according to the manufacturer’s instructions (eBioscience). Statistical Analysis Statistical analysis was performed with SPSS 13.0 software (Chicago, USA). Data are expressed as mean 6SD. One way- ANOVA was used to compare the differences between groups, followed by the Student-Newman-Keuls post-hoc analysis. Statis- tical significance was considered when p,0.05. Supporting Information Figure S1 Emphysema and airway remodeling in normoxia- and hypoxia-exposed mice. A, Representative H&E stained lung sections of the indicated genotypes showing alveoli and bronchiolar walls. Bars, 25 mm. B-C, Quantification of B, mean linear intercept (MLI), and C, alveolar area. n.s., not significant. (JPG) Figure S2 Treatment of hypoxia- and normoxia-ex- posed mice with Z62954982. A, Right ventricle hypertrophy assessed by ratio of (RV/LV+S) from the hearts of normoxic and hypoxic wt and abr2/2 mice treated with Z62954982 at 10 mg/ kg every other day or at 20 mg/kg daily for 3 wks. Control mice were administered i.p. with equal amount of vehicle. Bars, mean+SEM. n = 3–4 mice/group. *, p,0.05, data were analyzed by one way ANOVA. B–C, Western blot analysis of representative samples (B) and quantification (C) of activated Rac1 in mice treated with 10 mg/kg Z62954982. n = 3 samples/group. Lysates were generated 1 hr after injection of the drug. (TIF) Figure S3 Lack of toxicity of Z62954982. A, representative H&E-stained liver sections of the indicated mice treated with 10 mg/kg every second day. B, analysis of total bone marrow PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49756