We generated Stat5f/f;Alb-Cre mice by breeding Stat5f/f mice with Alb-Cre transgenic mice. Stat5f/f and Alb-Cre transgenic mice were on a mixed background. Only 10 to 18-week-old male mice were used in the experiments unless otherwise indicated. We treated the animals humanely and performed procedures according to the protocol approved by the Animal Use and Care Committee at the National Institute of Diabetes and Digestive and Kidney Diseases.
Liver Fibrosis Induced by CCl4
Hepatic fibrosis in mice was induced by intraperitoneal (i.p.) injection with 2 ml/kg body weight of 10% CCl4 (Sigma, St. Louis, MO) dissolved in olive oil (Sigma, St. Louis, MO), 3 times a week for 12 weeks. For growth hormone (GH) stimulation, mice were injected 2 μg/g body weight of GH by i.p. Four hours after injection they were sacrificed and livers were harvested for analyses.
Isolation of primary MEF cells
Primary MEFs were isolated from day E14.5 Stat5+/+ and Stat5−/−embryos by first mincing the embryos, then digesting in 0.05% trypsin/0.02% EDTA for 30 minutes at 37°C, pelleting the tissue, and resuspending in growth medium consisting of Dulbecco’s Modified Eagle medium (DMEM) with 10% FCS. MEFs were maintained in high-glucose DMEM supplemented with 15% FBS, 50 μg/ml streptomycin sulfate,50 units/ml penicillin G sodium, β-mercaptoethanol, and non-essential amino acid in an atmosphere of 5% CO2 at 37°C.
The retroviral-expression vector carrying a wild-type Stat5A gene was based on an MSCV-IRES-GFP backbone (gift from Richard Moriggl, Ludwig-Boltzmann Institute, Vienna, Austria). 293T cells were transfected with the plasmid using FuGENE (Roche, Indianapolis, IN). Supernatants were collected for 48–72 h after transfection and passed through a 0.45-μm filter before freezing at −80°C. For the infection, 106 Stat5−/− MEFs were seeded on a 10-cm culture dish and infected the next day with retrovirus in the presence of 8 μg/ml polybrene. After infection, non fluorescent cells and GFP-expressing cells were isolated using the FACS Vantage (Becton Dickinson, San Jose, CA) and sorted directly into PBS. Sorted MEFs were maintained in Dulbecco’s Modified Eagle medium (DMEM) supplemented as described above.
Affymetrix microarray analysis
Primary MEFs derived from Stat5+/+ or Stat5−/−embryos were cultured to passage 13. After starvation for 5 hours, MEFs were stimulated with GH (1 μg/ml) for 2 hours. Unstimulated samples were used as control. Total RNAs were prepared by using a RNeasy Plus Mini Kit (Qiagen, Valencia, CA). RNA quality was verified using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). Microarray analyses were performed using Affymetrix Mouse Genome 430 2.0 array GeneChips (Affymetrix, Santa Clara, CA). Microarray signals were analyzed using the Affymetrix RMA algorithm. Up- and down-regulated genes were selected based on P values less than .05 and fold changes of more than 1.5 or less than 1.5 as assessed by ANOVA using Partek Pro software (Partek, St Louis, MO). The microarray analyses were performed with 3 independent biologic sample sets. Microarray data have been deposited in Gene Expression Omnibus (GEO) (accession number: GSE21861).
Analysis of cell proliferation
The proliferation of cells was determined by a Trypan blue dye exclusion assay. In brief, primary Stat5+/+ and Stat5−/− MEFs (1 × 105 cells/well) were seeded on tissue culture plates and cultured in high-glucose DMEM. The number of viable cells was counted after 2, 4 and 6 days. MEFs were harvested with trypsin-EDTA. The cell suspension was loaded onto a hemocytometer (1:1) with the dye Trypan blue, which is taken up by dead cells. Both viable and dead cells were counted, from which both the percentage of dead cells and total cell number were calculated.
Primary Stat5+/+ and Stat5−/− MEFs were washed twice with PBS and fixed for 30 minutes at −20°C in 70% ethanol. Total DNA was stained with PI (5 μg/ml in PBS containing 50 μg/ml RNase A). Cell cycle distribution was determined by FACS analysis using a FACS Calibur (Becton Dickinson, San Jose, CA). Data are presented as a percentage of viable cells remaining in the respective cell cycle phase.
Antibodies, immunoblotting and immunostaining
In brief, primary MEFs were lysed by adding NuPAGE LDS Sample buffer (Invitrogen, Carlsbad, CA). Cell lysates were heat-denatured for 10 min at 70°C and loaded on a NuPAGE 10% Bis-Tris polyacrylamide gel. After electrophoresis in NuPAGE SDS running buffer using the Xcell SureLock Mini-Cell, proteins were transferred to a PVDF membrane according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The rabbit polyclonal anti-STAT5 (N-20), anti-STAT5 (C-17), anti-p15 (K-18), anti-p21 (C-19), anti-cyclin D1 (HD-11), anti-cyclin A (H-432), anti-cyclin B1 (H-433), anti-Cdk4 (C-22) and anti-βactin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used for probing western blots. Immunohistochemistry was performed using standard procedures. In short, liver tissues were removed and fixed in 10% neutral buffered formalin and embedded in paraffin wax. Five μm sections were prepared for hematoxylin and eosin (H&E) staining and immunofluorescence analyses. After deparaffinization, antigen unmasking was performed in a Decloaking chamber (Biocare Medical, San Diego, CA) using BORG Decloaker Solution (Biocare Medical, San Diego, CA) for 5 min at 125°C. The sections were blocked for 30 min in TBS-T containing 3% goat serum. Primary antibodies used in this study included rabbit anti-STAT5 (N-20), rabbit anti-phospho-STAT5 (Tyr694) (Cell Signaling Technology, Beverly, MA), rabbit anti-p15 (K-18) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-histone H3 (Ser-10) (Upstate Biotechnology, Lake Placid, NY), in addition to mouse anti-PCNA (DAKO Cytomation, Carpinteria, CA) and mouse anti-β-catenin (BD Transduction Laboratories, San Jose, CA). For double-labeling immunofluorescence analyses, sections exposed to a pair of primary antibodies were incubated in a 1:400 dilution of goat anti-rabbit IgG conjugated with a red fluorophore (Alexa Fluor 594; Molecular Probes, Eugene, OR) and goat anti-mouse IgG conjugated with a green fluorophore (Alexa Fluor 488; Molecular Probes, Eugene, OR) for 30 min at room temperature. Images were obtained with a Retiga Exi camera on a Olympus BX51 microscope (Olympus America, Center Valley, PA) using Image-Pro 5.1 software. For quantitation three images taken with the 40× objective were counted per mouse. Three mice from each experimental group were evaluated.
Cellular proteins were extracted from primary Stat5+/+ and Stat5−/− MEFs. Protein fractions were incubated overnight with an anti-CDK4 antibody and protein A–Sepharose beads at 4°C in RIPA buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 0.25% NP-40 and 0.5% sodium deoxycholate. The protein A–Sepharose-antibody-antigen complex was collected and washed three times with ice-cold RIPA buffer. The final pellet was resuspended with SDS-sample buffer and boiled for 5 min. This preparation was subjected to western blot analysis with the anti-p15 or anti-p21 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Chromatin immunoprecipitation assay
ChIP assay was performed as described previously.14
In brief, after starvation for 5 hours, primary Stat5+/+
MEFs were stimulated with GH for 45 minutes. Unstimulated samples were used as controls. MEFs were cross-linked in 1.5% formaldehyde for 15 min at 37°C. Cells were then harvested and sonicated using the Misonix Sonicator 3000 (Misonix, Farmingdale, NY, USA). Immunoprecipitation was carried out in TE buffer containing protease inhibitors (Sigma, St. Louis, MO). Chromatin was incubated with protein A Dynabeads (Invitrogen, Carlsbad, CA), which were pre-incubated with STAT5A or IgG antibody (R&D Systems, Minneapolis, MN, USA). Immunoprecipitated DNA was eluted and amplified by real-time PCR using a 7900 HT fast real-time PCR system (Applied Biosystems, Foster City, CA) and analyzed using SDS2.3 Software (Applied Biosystems, Foster City, CA). Sequence-specific primers used for amplification of the putative STAT5 binding sites (GAS sites) within the Socs2 and Cdkn2b
genes were as following: For the Socs2
GAS sequence, forward primer 5’-GGAGGGCGGAGTCGCAGGC-3’, reverse primer 5’-GACTTGGCAAGAGTTAACCGTC-3’; the primer sets for Cdkn2b
gene were: GAS1, forward 5’-GTTTTGCCGTGATGTCCTTG-3’, reverse 5’-ATCGCACTGCTTCGTGTAAC -3’; GAS2, forward 5’-GACAGGCATTGTCCAAGACA-3’, reverse 5’-GTGCCACATTCTCCCACTTT-3’.
RNA isolation and quantitative real-time PCR analysis
Total RNA was isolated from primary Stat5+/+ MEFs, Stat5−/− MEFs and liver tissues using Trizol reagent (Invitrogen, Carlsbad, CA). One μg amounts of RNA were reverse transcribed (cDNA reverse transcription kit; Applied Biosystems, Foster City, CA). Real-time quantification of mRNA transcript levels was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Real-time PCR was carried out using an ABI Prism 7900HT (Applied Biosystems, Foster City, CA). Individual PCRs were performed in triplicate on samples using GAPDH as a housekeeping gene. The primers used were: Cdkn2b, forward 5’-CCCTGCCACCCTTACCAGA-3’, reverse 5’-CAGATACCTCGCAATGTCACG-3’, yielding a 169 bp PCR product; Cdkn1a, forward 5’-GTGGCCTTGTCGCTGTCTT-3’, reverse 5’-GCGCTTGGAGTGATAGAAATCTG-3’, yielding a 126 bp PCR product; GAPDH, forward 5’-AACGACCCCTTCATTGAC-3’, reverse 5’ TCCACGACATACTCAGCAC-3’, yielding a 191bp PCR product.
All statistical analyses were performed using the Student’s t test (2 tailed, unpaired). A P value of 0.05 or less was considered significant.