Cell isolation and culture
Male Wistar rats (6 to 8 weeks old), which were obtained from the Centro de Bioterismo da Universidade Federal de Minas Gerais (CEBIO), were used for all of the studies. Rat inguinal adipose tissue was dissected into small pieces, digested with 0.15% collagenase B (Roche Applied Science, Indianapolis, IN, USA) in 0.1 M phosphate-buffered saline (PBS) for 40 minutes at 37°C. Mature adipocytes and connective tissues were separated from the cell pellet by centrifugation at 274.4 g for 10 minutes at room temperature. The cell pellet was resuspended in high-glucose Dulbecco modified Eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). The current protocol was adapted from a previously described protocol .
The initial passage of the primary cell culture was referred to as passage 0. We used cells in passage 3 only, except in the indicated experiments. The cells were maintained in complete media (DMEM + 10% FBS + penicillin/streptomycin) until they achieved 75% to 90% confluence. The cells were then replated in T75 tissue-culture flasks. The identity of the MSCs was defined by the criteria proposed by the International Society for Cellular Therapy . The experiments were approved by the local ethical committee on animal experimentation (CETEA/UFMG; protocol number 239/10).
Phenotypic analysis with flow cytometry
MSCs were recognized by using specific antibodies against CD54, CD73, and CD90. In addition, we assessed the lack of expression of the CD45 hematopoietic marker. All antibodies were purchased from Becton Dickinson (BD) Biosciences (San Jose, CA, USA). The secondary antibodies used were conjugated to Alexa Fluor 488 (Life Technologies).
For immunophenotypic analysis, the MSCs were detached by incubation with 0.05% trypsin-EDTA (Life Technologies) for 5 minutes, immediately resuspended in DMEM plus 10% FBS, and plated in 96-well culture plates at a density of 2 × 105 cells/well. Next, the cells were washed with PBS, and the cell suspension (100 μl) was incubated at 4°C for 30 minutes with the primary antibody (1:50) or with an isotype antibody (1:50) as a negative control. Then, the plate was centrifuged at 274.4 g for 10 minutes at 10°C. After two washes with PBS, the cell suspension was incubated at 4°C for 30 minutes with the secondary antibody (1:200). Subsequently, the cells were washed with PBS and analyzed with FACScan flow cytometry by using CELLQuest software (BD Biosciences). All fluorescence-activated cell-sorting (FACS) data were analyzed by using FlowJo software (Tree Star, San Carlos, CA, USA).
Adenoviral constructs encoding DsRed (Ad-DsRed) and the PV-DsRed fusion protein that was targeted to the cytosol (Ad-PV-NES-DsRed) were obtained as previously described . The adenoviruses were amplified in HEK-293 cells, a cell line that constitutively expresses the E1 protein. Both viruses (Ad-DsRed and Ad-PV-NES-DsRed) were generated by using standard procedures and stored at −80°C in PBS plus 7% glycerol. Stocks of the adenoviral particles were quantified as plaque-forming units (pfus) by using plaque assays in HEK-293 cells. On day 1 of culture, each adenovirus construct (at a multiplicity of infection (MOI) of 100) was diluted in regular culture medium (DMEM) containing 10% FBS. The experiments were performed at 36 to 48 hours, when approximately 70% of the cells were transduced.
MSCs were cultured in 96-well culture plates at a density of 1 × 104 cells/well and then transduced with the adenoviral constructs. After 24 hours, the cells were synchronized to G0 by serum starvation overnight. The next morning, the cells were washed in PBS and resuspended in HEPES buffer containing 3 μM Fluo-4-AM (Life Technologies) for 30 minutes at 37°C. Next, the cells were washed, resuspended in HEPES, and then stimulated with HEPES containing 10% FBS. The resultant fluorescent signal was monitored for the whole well over time by using a Synergy 2 multimode plate reader (BioTek, Winooski, VT, USA). The wavelength for excitation was 485/20 nm; emission was measured at 530/25 nm, and the dichroic that was used was the top 50%. The experiments were performed at 37°C. The mean peaks of the maximum and minimum fluorescence were analyzed by using Excel and plotted by using GraphPad software (La Jolla, CA, USA).
Analysis of cell viability
To assess the cell viability of the MSCs, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Life Technologies), as previously described . In brief, the MSCs were plated in 24-well culture plates at a density of 1 × 105 cells/cm2 per well. MTT (5 mg/ml) was added to each well of the monolayer cultures, and the cultures were incubated in a humidified 5% CO2 incubator at 37°C. Two hours later, the cell morphologies and formazan salts were visualized by using an inverted optical microscope. The formazan salts were dissolved with 10% SDS-HCl overnight, and the optical density was measured at 595 nm by using a microplate reader (Elx800; BioTek). Staurosporine was used as a positive control for cell death at a concentration of 100 nM in DMEM containing 10% FBS. The values from the samples are expressed as percentages of the control. All data were analyzed by using GraphPad software.
Analysis of cell death by flow cytometry
A flow-cytometric DNA fragmentation assay was used as a quantitative measure of cell death . Forty-eight hours after transducing the MSCs with the adenoviral constructs, the cells were collected by centrifugation and lysed with 300 μl of a hypotonic solution containing 0.1% sodium citrate, 0.1% Triton X-100, and 20 μg/ml propidium iodide (PI; Life Technologies). Then the cells were incubated at room temperature for 1 hour and analyzed with a FACScan flow cytometer (BD Biosciences) for shifts in PI fluorescence, which are indicative of nuclei with hypodiploid DNA content. All data were analyzed by using FlowJo software (Tree Star).
Mitotic index measurements by immunofluorescence
Confocal immunofluorescence was performed as previously described [10, 16]. In brief, the cells were labeled with an anti-phospho-histone-3 monoclonal antibody (1:500; Millipore, Billerica, MA, USA) and then incubated with an Alexa Fluor 488-conjugated secondary antibody (1:1,000; Life Technologies) and the Hoechst nuclear stain (200 ng/ml; Life Technologies). Images were captured by using a Zeiss LSM 510 confocal microscope with a 63×/1.4 NA objective lens. The samples were excited at 488 nm and observed at 505 to 550 nm to detect Alexa Fluor 488, excited at 405 nm, and observed at 420 to 460 nm to detect Hoechst staining. Mitotic indexes were scored for the cells in each phase of mitosis according to the phospho-histone-3 distribution and DNA condensation pattern .
Immunoblotting was performed by using standard methods [10, 16]. In brief, the cells, which were grown in T75 tissue-culture flasks, were washed 3 times with PBS and lysed in buffer containing 150 mM NaCl, 1 mM EDTA, 20 mM Tris–HCl, 0.5% Nonidet P-40, and a protease-inhibitor mixture (Sigma-Aldrich). The protein concentration was determined spectrophotometrically by using the Bradford method (Sigma-Aldrich), and 40 μg of protein was separated by electrophoresis in a 4% to 15% polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS) plus 0.5% Tween 20 (TBST) for 60 minutes and then incubated with primary antibodies against total Erk1/2 or phospho-Erk1/2 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), cyclins A, B1, D1, D2, D3, E, or E2 (1:500 to 1,000, Cell Signaling Technology) or GAPDH (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The incubations with the primary antibodies were carried out overnight. After three 5-minute washes with TBST, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000) (Sigma-Aldrich) for 1 hour at room temperature. After three additional 5-minute washes with TBST, the membranes were developed by using enhanced chemiluminescence (ECL Plus; GE Healthcare Life Sciences, Piscataway, NJ, USA). Subsequently, the films were scanned and analyzed by using Image J software.
All of the data shown represent at least three independent experiments and are expressed as the mean ± standard error of the mean (SEM). The statistical analyses were performed with GraphPad software. The experiments with more than two data groups were compared by using one-way ANOVA and the Bonferroni posttest. P values of <0.05 were considered to be statistically significant.