Together these and results establish the anti-fibrogenic role of BMPR2/Smad1/5/8 signaling in the pancreas CP model. While more severe fibrosis following CP induction was observed in BMPR2 deficient mice versus their wild-type Levonorgestrel counterparts, neither persistent endocrine dysfunction nor overt diabetes was observed in either group. pancreatic fibrosis, PSC activation and leukocyte infiltration after CP induction compared to wild-type mice (Model of Chronic Pancreatitis CP was induced in the mice by repetitive injections of cerulein [21]. Briefly, the mice were subjected to cerulein injection intraperitoneally (50 g/kg, 5 hourly injections/day, 3 days/week) for 4 weeks. Normal saline was given at the same volume and frequency in control mice. The mice were euthanized at day 4 after completion of cerulein or normal saline injections. The pancreas was harvested for histological analysis and snap frozen for protein and RNA preparation. Morphological Examination Pancreatic tissue samples were fixed in 10% formalin Levonorgestrel and subsequently embedded in paraffin. Five m thick sections were prepared for hematoxylin-eosin (H&E) staining (Vector Laboratories, Inc., Burlingame, CA) and immunohistochemistry analysis. Quantitative Analysis of Pancreatic Fibrosis Sirius red staining was performed Rabbit Polyclonal to eNOS (phospho-Ser615) on pancreas paraffin sections for the quantification of total intra-pancreatic collagen deposition [22]. Ten to fifteen images were taken from nonoverlapping fields of each section. Sirius red-stained area was quantified using NIS-Elements Br 3.0 imaging analysis software and expressed as percentage of the total area measured. Immunohistochemistry Analysis The pancreatic paraffin sections were prepared for immunohistochemistry analysis of -SMA and CD45 with ABC kit and DAB kit (Vector Laboratories, Inc. Burlingame, CA, USA) according to the manufacturers instructions as described previously [21]. Briefly, following deparaffinization and hydration, antigen retrieval, blockage of endogenous peroxidase activity and nonspecific protein binding sites, the sections were then incubated with rabbit antibodies against -SMA and CD45 overnight at 4C. The sections were subsequently incubated with a biotinylated anti-rabbit antibody for one hour at room temperature, and then with ABC reagents for 30 minutes at room temperature. Finally the sections were stained using DAB kit followed by hematoxylin nuclei counterstaining and dehydrated, mounted with a permanent mounting solution (Vector Laboratories, Inc., Burlingame, CA, USA). Immunofluorescence The frozen pancreatic tissue sections were dried for 60 minutes at room temperature, and then fixed with methanol at ?20C for 20 minutes. The PSCs were fixed with 4% paraformaldehyde for 15 minutes. Immunofluorescence was performed using TGF-1, BMPR2 and FN antibodies as described before [21]. Isolation and Culture of Mouse PSCs Mouse primary PSCs were isolated from the pancreas of BMPR2+/? and wild-type mice by an outgrowth method [23]. The cells were cultured in Dulbeccos modified essential medium (DMEM, Mediatech Inc., Manassas, VA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies-Invitrogen, Grand Levonorgestrel Island, NY, USA) at 37C in a humidified incubator (made up of 95% of O2 and 5% of CO2). Passages 1 to 3 were used. Western Blotting Protein lysates were prepared from mouse pancreatic tissue samples or cells in 1X lysis buffer (Cell Signaling Technology, Inc., Billerica, MA, USA). Protein concentrations were measured using a protein assay dye (Bio-Rad Laboratories, Hercules, CA, USA). Western Levonorgestrel blotting analysis was performed as described previously [24]. Quantitative Polymerase Chain Reaction (qPCR) Total RNA was extracted from pancreatic tissue samples of the mice, and reversely transcribed to cDNA using RETROscript kit (Life Technology Co., Grand Island, NY, USA). qPCR was performed using Levonorgestrel Taqman gene expression master mix and specific gene probe sets as previously described [24]. The probe sets of mouse BMP2 (Mm013401798_m1), BMP4 (Mm00432087_m1), BMP7 (Mm00432102_m1), BMPR1a (Mm00477650_m1), BMPR2 (Mm00432134_m1), TGF-1 (Mm01178820_m1), and 18s (Hs99999901_s1) (Life Technology Co., Grand Island, NY, USA) were used in the study. The signals acquired from BMPs, BMPRs, and TGF-1 were normalized to the signals acquired from 18s, and expressed as fold of control. Statistical Analysis Data were expressed as means SEM. experiments were repeated 2C3 times and similar results were obtained. Differences between two groups were analyzed using the Students in response to BMP2 stimulation, pSmad1/5/8 level was elevated in PSCs isolated from wild-type mice, and this effect was abolished in PSCs from BMPR2+/? mice (Physique 6). Subsequently, the PSCs from BMPR2+/? mice lost BMP2s inhibitory effect on TGF–induced ECM production (Physique 7). Together these and results establish the.