Cell culture and generation of HBE cells with partially depleted mtDNA ρ− cells
The immortalized human bronchial epithelial cell line (HBE) was a gift from Professor Wen Cheng (School of Public Health, Zhongshan University). The parental HBE cells (ρ+) were maintained in growth medium containing a 4.5 g/L glucose DMEM, 2 mmol/L L-glutamine, 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/ml streptomycin. The partially depleted mtDNA cells (ρ−) were generated by treatment of ρ+ HBE cells with 50 ng/ml ethidium bromide (EB) in growth medium supplemented with 50 μg/ml uridine (Sigma) and 100 μg/ml sodium pyruvate. The ρ− cells were cultured using the growth medium plus 12.5 ng/ml EB and 50 μg/ml uridine, which provides an alternative source of energy through glycolysis to ensure optimal growth. Cell viability was assessed by the MTT assay and found to be 85%.
Determination of mtDNA copies
The mitochondrial DNA (mtDNA) copy number was tested by quantitative real time PCR using the 2−ΔΔCt
method with GAPDH as a reference as described previously (Evdokimovsky et al., 2011
). Using the ρ+ HBE cells as the standard, whose mtDNA copy number (mtDNA amount / nDNA amount) was defined as one, the relative mtDNA copy numbers of the ρ− cells were calculated. ND1 and 16S rRNA was expressed as mean mtDNA, while 18S rRNA was expressed as mean nDNA. Briefly, total cell DNA was extracted with E.Z.N.A.® Tissue DNA Kit (OMEGA, USA) and quantified by spectrometry. Fifty ng DNA was used to amplify the primers () using SYBR Green detection on an 7500 Real-time PCR System (Applied Biosystem, Carlsbad, CA, USA). All reactions were conducted in triplicate in 96-well MicroAmp® optical tubes (Applied Biosystems, USA). PCR conditions were set as follows: an initial step of 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C for 30 sec, 55 °C for 30 sec, and 72 °C.
Mitochondrial DNA primer sequence
Radon and its progeny Irradiation
Exponentially growing ρ− and ρ+ HBE cells were plated onto Transwell membrane (Corning, USA) with a 6 μm mylar bottom at a density of 1 × 105 cells 2 days before irradiation. Cells were placed in a gas inhalation chamber (Chinese Academy of Military Medical Sciences, Beijing, China). The gas chamber was connected with a multifunctional radon chamber purchased from Donghua University in China. Randon and its progeny was produced by a radium source using a Changhe pump machine (model BT00-300, China) and pumped into the gas chamber. Cells were directly exposed to radon and its progeny at concentration of 20, 000 Bq/m3 for 20 min every other day. During exposure, the cells were kept at 37 °C in medium bath. This procedure was continued for 10 passages and each passage was exposed twice. The optimal time and concentration were determined by MTT assay.
Cell staining with fluorescent probe
All cell staining was performed with cells grown in 35 mm dish with 0.17 mm glass bottom. mtDNA staining was achieved by diluting stock PicoGreen solution at 3 μl/ml directly into cell culture medium. (Molecular Probes Inc., Oregon, USA), which specifically visualizes only mitochondrial nucleic acid. The cells were then incubated for 1 hr, and washed twice with medium. Mitochondrion of cells were co-stained with mitochondrion selective dye Mitotracker Red CM-H2XRos (Molecular Probes) by adding 50 nM directly to the culture medium and incubating cells for 30 min.. The cells were then rinsed thrice in pre-warmed PBS and visualized with living cell system (Cell^ R, Olympus, Japan).
Apoptosis analysis with flow cytometry
Apoptosis was determined using annexin V-FITC/propidium iodide (PI) staining assay with an apoptosis assay kit (Invitrogen, USA). Cells with confluence of 70-80% were trypsinized and stained for apoptosis detection through FACS5000 flow cytometry (BECKMAN, CA, USA) according to the manufacturer’s instructions. For analysis, early apoptotic cells showed a positive staining of annexin V-FITC. Late apoptotic and dead cells display both membrane positive staining with annexin V-FITC and nuclear positive staining with PI.
Measurement of mitochondrial transmembrane potential
Mitochondrial transmembrane potential (MMP) was investigated using JC-1 (5,5V,6,6V -tetrachloro-1,1V,3,3V-tetraethylbenzimidazolyl-carbocyanine iodide), which was reported to provide more accurate estimates of MMP than other fluorescent dyes (Mathur et al., 2000
). This dye accumulates in the mitochondrial matrix under the influence of MMP, where it reversibly forms monomers (green) and J aggregates (red) with characteristic absorption and emission spectra (Reers et al., 1995
cells were incubated with JC-1 (5 μg/ml final concentration) for 1 hr at 4°C, washed, and immediately analyzed by flow cytometry using 488 as excitation and 530 or 590 nm as emission wavelengths. Bivariate plots of FL1 versus FL2 were used to analyze MMP (Smiley et al, 1991
) Flow cytometric data presented are representative of three experiments. The ratio of fluorescent identity at FL2/FL1 was considered as the relative MMP value.
Measurement of intracellular ROS levels
Intracellular reactive oxygen species (ROS) levels were measured by a cell-permeating probe CM-H2DCFDA (Invitrogen, USA). CM-H2DCFDA is a non-polar compound which is hydrolyzed after entering the cell to form a non-fluorescent derivative, subsequently converted into a fluorescent product in the presence of an oxidant. Briefly, cells were washed, and then loaded with 10 μM CM-H2DCFDA for 1 hr. Excitation was set at 488 nm, and emission was recorded on an FL1 detector (525 nm). Data presented here are representative of three experiments. The fluorescent intensity was taken as the relative intracellular ROS level.
Cell cycle analysis
The cells were harvested with a trypsin-EDTA solution to produce a single-cell suspension, pelleted by centrifugation at 600 g for 10 min, and washed with PBS. The cells were then resuspended in a solution of PBS (pH 7.4) containing 25 mg/ml PI (Sigma Chemical Co., St. Louis, MO, USA), 0.1 mM EDTA and 0.01 mg/ml DNase-free RNase. The samples were incubated for 15 min at room temperature and followed with analysis on a FACS5000 flow cytometer.
Data are presented as mean ± standard deviations. Data were subjected to ANOVA and comparisons between irradiation groups (radon) and controls were determined by the Student’s t-test. A p <0.05 was considered to be significant.