Cell lines and culture
Human prostatic carcinoma cell lines, including PC3, DU145 cells, and bone marrow-derived MSCs were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. PC3, DU145 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). MSCs were cultured in MSCs basal medium (all from Invitrogen, Carlsbad, CA, USA). MSCs were transfected with the adenoviral vector GFP-mock (Invitrogen). After transfection about 48 hours, MSCs-GFP were collected for further experiments. All cells were cultured at 37 °C in a 5% CO2 humidified atmosphere.
In vivo xenograft experiment
Nude mice, 6–8 weeks old, were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China, and housed in pathogen-free conditions. All aspects of the animal care and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Chinese Academy of Sciences’ Committee on Animals. PCa cells were prepared as single-cell type suspensions (1 × 106 cells in 200µL PBS) and subcutaneously administrated in the armpit area of nude mice. When tumors grew to approximate 200 mm3 size, nude mice were randomly divided into three groups: with solvent or docetaxel treatment (15 mg/kg/week via intraperitoneal injection, 5 mice per group). With that, mice were injected with the green fluorescent protein (GFP)-labeled MSCs (MSCs-GFP) through tail vein every 3 days. Mice were examined every day and tumor growth was evaluated by measuring the length and width of tumor mass. All tumor-bearing mice survived until they were sacrificed at the end of the experiment, then tumors were removed and dissected quickly for frozen section preparation, while others were stored at -80℃.
Cell apoptosis assay
Cells (2 × 105 cells/well) were cultured in 6-well plates to 70–80% confluence. The cells were then treated with docetaxel for 48 hours. PI/Annexin V-FITC assay was used to measure apoptotic cells by flow cytometry according to the manufacturer’s instruction (Keygen Biotech. Co., Ltd, Nanjing, China, Cat.KGA108). The cells were collected by trypsinization and were washed with ice cold phosphate buffered saline (PBS). Cells were then incubated in 300 µL of 1 × binding buffer containing 5 µL Annexin V and 5 µL PI for 30 min at room temperature in the dark. Apoptosis of cells was measured on a BD FACScan flow cytometer (BD Biosciences). At least 30,000 gated events were acquired from each sample. Results are expressed as the percentage of apoptotic cells (PI and Annexin V positive) in the gated cell population.
Cells were plated at a density of 2 × 105 cells per well in 6-well plates cocultured with MSCs and treated with docetaxel (20 µM) for 48 h. CCK8 test was performed to evaluate the extent of cell proliferation (OD values) according to the manufacturer’s instructions.
Real-time quantitative PCR (RT-PCR)
To quantify mRNA expression of PCNA, caspase-3 and TGF-β1, total RNA was isolated using Trizol reagent (Invitrogen) and cDNA synthesis was performed using the Prime Script RT reagent Kit (Takara, Kyoto, Japan) according to the manufacturer’s specifications. Quantitative PCR was performed using SYBR Green PCR Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions. β-actin was used as an internal control for RNA integrity and loading normalization.
Western blot analysis
Cells were lysed in RIPA lysis buffer (Beyotime) with 1 mM PMSF. Equal amount of protein was separated by SDS-PAGE and transferred to NC membrane. The membranes were washed, blocked and incubated with specific primary anti-human antibodies against p62/SQSTM1 (all from Cell Signaling Technology, Inc., Danvers, MA, USA), LC3 (Novus Biologicals, Littleton, CO), TGF-β1 and β-actin (Abcam, Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Hangzhou HuaAn Biotech). Signals were visualized by chemiluminescent detection (Beyotime).
Enzyme linked immunosorbent assay (ELISA)
ELISA assays were performed using commercial ELISA kits (R&D Systems, Minneapolis, MN) according to manufacturer instructions. Assays were performed in duplicates, and readings were compared with standard curves obtained with standard protein provided with the kit. Means and standard deviations of concentrations in triplicate samples were compared by t-test.
Cells (1 × 106) growing to 50%-60% confluence in 10 cm petri dishes were transfected with TGF-β1 siRNA sequences or their corresponding mock sequences using a Lipofectamine 2000 kit (Invitrogen, Cat.11668-019) with the procedure provided by the manufacturer. Cells were observed under a fluorescence microscope and harvested 48 h after transfection.
Fugene HD transfection reagent (Calbiochem, La Jolla, CA) was used to transfect cells with GFP-LC3 expressing plasmids according to the manufacturer’s instructions. After initial treatment, autophagy was detected by counting the number of GFP-LC3-positive dots per cell under fluorescence microscope (Olympus IX71).
Electron microscopic analysis
Cells were fixed in 2.5% glutaraldehyde in PBS (pH = 7.4) for 2 hours at room temperature, then postfixed in 1% osmium tetroxide in water for 1 hour, dehydrated in an ascending series of ethanol, and at last embedded in araldite (Basel, Switzerland). After solidified, 50 nm sections were cut on a LKB-I ultramicrotome and picked up on copper grids, post-stained with uranyl acetate and lead citrate, and observed in a Philips CM-120 TEM.
All of the experiments were repeated at least three times. Final data were expressed as mean ± standard deviation (SD). Statistical analysis of the data was done by using GraphPad Prism 5. Student’s t-test was used to compare between mean values of two groups. Value of at least p < 0.05 was considered statistically significant.