Antibodies and reagents
Antibodies used in this study are as follow: monoclonal rabbit anti-BST-2(Abcam, 1:1000), polyclonal rabbit anti-Bcl-2 (CST, 1:1000), polyclonal rabbit anti-Caspase-3 (CTS, 1:1000), monoclonal mouse anti-Caspase-8(1C12)(CST, 1:1000), polyclonal rabbit anti-Caspase-9 (CST, 1:1000), polyclonal rabbit anti-LC3A/B(CST, 1:1000), polyclonal rabbit anti-AIF (CST, 1:1000), polyclonal rabbit anti-COX IV (CST, 1:1000), monoclonal mouse anti-Lamin A/C(4C11) (CST, 1:1000), monoclonal mouse anti-cytochrome C (Beyotime Biotech, 1:200), polyclonal mouse anti-β-Actin and anti-GAPDH (Santa Cruz Biotechnology, 1:8000), HRP-labeled goat anti-mouse and anti-rabbit IgG (Earthox, 1:10000).
DMEM medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from HyClone (Logan, USA). L-glutamine was purchased from Gibico (CA, USA). Annexin V-FITC/PI apoptosis detection kit and was purchased from TransGen Biotech (Beijing, China). 7-AAD viability staining solution was purchased from BioLegend (San Diego, CA, USA). Nuclear extraction kit and mitochondria extraction kit were obtained from Pierce Biotechnology (Rockford, USA). Ac-DEVD-CHO was purchased from Beyotime Biotech (Nanjing, China). CD317-specific siRNA (named as siR317) and Normal Control siRNA (name as NC) were synthesized by GenePharma (Shanghai, China). The sequences of the siRNA targeting human CD317 were 5-CCAGGUCUUAAGCGUGAGAdTdT-3 and 5-UCGCGGACAAGAAGUACUAdTdT-3 (corresponding to base pairs 432–450 and 452–470 of the human CD317 sequence, respectively) , and the sequences of murine CD317-specific siRNA were 5-GGGUUACCUUAGUCAUCCUdTdT-3 and 5-GCUUGAGAAUGAAGUCACGdTdT-3 (corresponding to base pairs 126–144 and 379–397 of the murine CD317 sequence, respectively), the NC siRNA (5-UUCUCCGAACGUGUCACGUdTdT-3) was used as negative control. MigR1-CD317 plasmid (named as plasCD317) was constructed in our lab. Briefly, the full length of human CD317 CDS was cloned from Jurkat cells by RT-PCR using specific primers, digested with Bgl II and Xho I, then subcloned into the expression vector MigR1 and sequenced.
Cells and transfection
Hela (an epithelial cell line from female cervical cancer), SK-OV-3(a human ovarian cancer cell line), MCF-7 (a luminal human breast cancer cell line), HepG2 cells(a hepatocellular carcinoma cell line), sp2/0 cells (a mouse myeloma cell line), U266(a human myeloma cell line) and HEK293T (a CD317 negative human embryonic kidney cell line) were obtained from ATCC or Cell bank, Chinese academy of sciences(Shanghai, China) and routinely culture in DMED medium or RPMI-1640 medium supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 10 mg/mL streptomycin. All cultures were maintained in a humidified 5 % CO2 incubator at 37 °C, and routinely passaged when 80–90 % confluent.
The CD317-positive cells were transfected with NC (named as NC cells) or siR317 (named as siR317 cells), while the HEK293T cells were transfected with MigR1 or plasCD317, respectively. All transfections were performed using Lipofectamine 3000 (Invitrogen, USA) or Gene Pulser Xcell (BioRad, USA) according to the manufacturer’s instructions.
After the transfection, cells were cultured in normal or serum deprivation condition for subsequent experiments.
FACS analysis for apoptosis
After transfection, cells were cultured in normal or serum withdrawal condition for another 48 h and then stained with 7-AAD or double-stained with Annexin V-FITC and propidium iodide (PI) using an apoptosis detection kit for flow cytometry analysis. All samples were analyzed using flow cytometer (FACS Canto II, BD).
JC-1 assay for MMP
Mitochondrial membrane potential (MMP,ΔΨm) was evaluated by JC-1 reduction according to the manufacturer’s instructions. After indicated treatment and wash, cells were incubated with 2.5 μM JC-1 at 37 °C for 30 min and then quantified using flow cytometer (FACS Canto II, BD).
Immunofluorescence and microscopy
Hela cells mounted on glass slides were fixed with 4 % paraformaldehyde (PFA) for 20 min at room temperature and then permeabilized with 0.1 % Triton X-100 for another 20 min. Cells were blocked with goat serum for 20 min at room temperature and then incubated with primary antibody (polyclonal rabbit anti-AIF, 1:1000) at 4 °C. After overnight incubation, cells were further stained with rhodamine-conjugated goat anti-rabbit IgG (Molecular Probes), and 4, 6-diamino-2-phenylindole (DAPI, Roche) and analyzed on an optical microscopy (Olympus IX71, Tokyo, Japan). 10–15 high-powered fields were evaluated, and fixed fluorescence images were analyzed by Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).
Cytoplasmic and nuclear protein extraction
Cell cytoplasm and nucleus protein were collected with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). In brief, cells were washed with cold PBS and fully suspended in ice-cold CER I by vortex on the highest setting for 15 s, followed by incubation on ice for 10 min. Homogenates were added ice-cold CER II, incubated on ice for 1 min and then centrifuged at 16,000 g for 5 min at 4 °C. The supernatants (cytosol) were collected and the pellets were suspended in ice-cold NER and incubated on ice for 40 min, vortex for 15 s every 10 min. The lysates were again centrifuged at 16,000 g for 5 min, and the supernatants (nucleus) were collected. The distributions of protein in cytosol and nucleus were analyzed by western blot.
Isolation of mitochondria was performed using mitochondria isolation kit (Pierce). Briefly, 1 × 107 cells were suspended in 800 μL Reagent A and incubated on ice for exactly 2 min, then were added 10 μL Reagent B followed with another incubation on ice for 5 min. After that, 800 μL Reagent C was added and then the mixture was centrifuged at 700 g for 10 min at 4 °C. The supernatant was transferred to a new 2.0 mL tube and centrifuged at 12,000 g for 15 min at 4 °C to obtain the mitochondria. At last, the isolated mitochondria were lysed in RIPA buffer containing 1.0 mM PMSF (Beyotime, China) to extract protein for further analysis.
Hela cells were cultured in normal or serum-deprivation condition for 24 h, cell lysates were prepared and immunoprecipitated with anti-CD317 or control Ig. The precipitates and lysates were subjected to Western blotting with antibodies for the indicated antigens.
Cells were washed with PBS and lysed with RIPA buffer supplemented with 1.0 mM PMSF. After 15-min incubation on ice, lysates were centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was collected, and the protein concentration was measured by bicinchoninic acid (BCA) assay (Beyotime, China). Whole cell lysates (25–50 μg of denatured protein) were separated on a 4–20 % gradient SDS polyacrylamide gel and then transferred to a PVDF membrane (Millipore). The membrane was blocked for 1 h at room temperature in PBST, pH 7.4 (PBS with 0.1 % Tween-20) with 5 % BSA. After washing, the membrane was incubated with primary antibodies overnight at 4 °C, followed by extensive washing and incubation with HRP-conjugated secondary antibodies. At last, protein was visualized by an enhanced chemiluminescense detection kit (Millipore, USA). After stripping, the blot was reprobed with β-Actin or GAPDH antibody (Santa Cruz) to ensure equal loading of total protein.
All data represent at least 3 independent experiments and are expressed as mean ± SEM. Statistical comparisons were made using Student’s t-test. P < 0.05 was considered statistically significant.