All Rem2 constructs to be used for imaging were subcloned from pCMV-HA into pECFP, pEYFP or pEGFP

er was determined by hemacytometer and differential cell counts were performed on Cytospin-prepared slides stained with Richard Allen three-step stain. Left lungs were frozen immediately for later biochemical analysis. A lobe of the right lung was stored in RNAlater for isolation of mRNA and remaining right lung lobes were flash frozen in liquid nitrogen for the analysis of hydroxyproline or SDS-PAGE. Fibrosis model After performing the inflammation studies, we obtained a new lot of bleomycin from a different supplier and determined through a pilot AMI-1 experiment that the bleomycin dose needed to be reduced. Consequently the fibrosis experiments were performed using 1.5U/Kg of bleomycin. With the reduction in bleomycin dose we also reduced the amount of CDDO-Me used. Two independent experiments were performed 9570468 using 300 ng CDDO-Me in one experiment and 350 ng CDDO-Me in the other. To assess the results, a general linear model employing a two-way analysis of variance with interaction was developed to evaluate the effect of treatment and CDDO-Me dosage. Mice received 1.5U/kg of bleomycin in a 40 ml volume of PBS by OA on day 0, and were treated with either 300 ng or 350 ng CDDO-Me in 40 ml volume by OA every other day from day -1 to day 9. Control groups received bleomycin plus vehicle. Fibrosis was assessed on day 21. The mice were anesthetized and euthanized as described. The lungs were removed without lavaging. The left lung was inflated in 10% neutral buffered formalin at 25 cm water pressure, and used for histology. A lobe of the right lung was stored in RNAlater for isolation of mRNA and remaining right lung lobes were flash frozen in liquid nitrogen for the analysis of hydroxyproline or SDS-PAGE. Materials and Methods Ethics Statement All animal procedures were approved and supervised by the University of Rochester University Committee on Animal Resources. Prior to surgical isolation of the lungs for analysis, mice were anesthetized with an I.P. injection of avertin, followed by exsanguination; all efforts were made to minimize suffering. Primary human lung fibroblasts were derived from anatomically normal areas of lung tissues obtained from patients undergoing wedge biopsy for other medical reasons and excess tissue was obtained. All tissue donors provided informed written consent as described in study protocols approved by the University of Rochester Institutional Review Board and conforming to the Helsinki Declarations. Animals Male C57BL/6J mice age 810 weeks were obtained from The Jackson Laboratory and housed at the University of Rochester. The mice were handled and maintained using microisolation techniques 12187403 with daily veterinarian monitoring. All experiments were conducted under a protocol approved by the institutional animal care use committee of the University of Rochester Medical Center. The number of mice used for each analysis is shown in the figure legends. ELISA Inflammatory cytokines were measured in lung homogenates as described. Briefly, lungs were homogenized in Buffer A, centrifuged, and the clarified homogenate was assayed for IL-6, KC and TGFb using commercial ELISA kits according to the manufacturer’s directions. To measure total TGFb, samples were acid activated as directed by the manufacturer to release latent TGFb Inflammation model Bleomycin was diluted in PBS and 2 U/kg was administered in a volume of 40 ml by CDDO-Me Inhibits Pulmonary Fibrosis/Inflammation in the sample. Western blots Hydroxyproline assay Hy