Cell culture and transfection
MDA MB 231 and HBL100 cells were procured from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics. The cultures were incubated in a humidified atmosphere at 37 °C with 5% CO2. Endogenously expressed MUC16 was stably knocked down using a MUC16 shRNA construct (pSUPER-Retro-MUC16-sh—provided by Dr Ilene K Gipson from Harvard Medical School) in MDA MB 231 and HBL 100 breast cancer cells by stable transfection method. Using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), the MUC16 shRNA and scramble vector were transfected into phoenix cells, a packaging cell line that produces high-titer retrovirus in culture. MDA MB 231 and HBL 100 cells were then seeded in 24-well plates at 2 × 104 cells per well and grown to 40% confluence in serum-free growth medium. Tissue culture medium from transfected phoenix cells was filtered 48 h after transfection, and the viral supernatant was used to infect the cultures of subconfluent MDA MB 231 and HBL 100 cells after the addition of 4 mg/ml polybrene. Isolated clones were obtained using antibiotic selection (puromycin 3 μg/ml) and were further expanded to confluent levels to obtain stably transfected cells.
For immunoblotting, MDA MB 231 and HBL 100 cells were lysed in RIPA buffer (50mM Tris–HCl, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease inhibitors (1mM phenyl–methyl sulphonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin). Samples were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinyldifluoride membranes (Millipore Coporation, Bedford, MA, USA) for immunodetection. After quick washing in PBST (phosphate-buffered saline (PBS) and 0.1% Tween 20), the membranes were blocked in 5% non-fat dry milk in PBS for at least 1 h and then incubated with primary antibodies MUC16 (mouse, 1:4000, DAKO, Carpinteria, CA, USA), JAK2 (Rabbit, 1:1000), pSTAT3 (Tyr705) (mouse), c-Jun (rabbit, 1:2000), Cyclin D1 (rabbit, 1:1500), Cyclin E (mouse, 1:1500), Cyclin A (rabbit, 1:1500), Cyclin B1 (rabbit, 1:2000), phospho-Aurora kinase A (rabbit, 1:2000), Bcl-2 (mouse, 1:1500), TRAIL (rabbit, 1:2000) JNK (rabbit, 1:2000), pJNK (rabbit, 1:2000), caspase 3 (mouse, 1:2000), caspase 9 (rabbit, 1:2000) and anti-β-actin (mouse) (diluted in 2% bovine serum albumin in PBS) overnight at 4 °C. Then the membranes were washed (3 × 10min) in PBST at room temperature and probed with the appropriate secondary antibodies at 1:2000 dilutions for 1 h at room temperature and washed 3 × 10min with PBST. The signal was detected with the ECL chemiluminescence kit (Amersham Bioscience, Amersham Place Little Chalfont, Buckinghamshire, UK).
Equal amounts of protein (500 μg) were incubated overnight with anti-MUC16, anti-Jak2, MUC1 (mouse monoclonal) and MUC4 (mouse monoclonal) antibodies in a 500 μl total volume. Protein A+ G-Sepharose beads (Sigma-Aldrich Corp., St Louis, MO, USA) were added to the lysate-antibody mix and incubated on a rotating platform for 3 h at 4 °C and then washed four times with lysis buffer. The immunoprecipitates or total cell lysates were electrophoretically resolved on SDS–polyacrylamide gel electrophoresis (8%). Resolved proteins were transferred onto the polyvinyldifluoride membrane. After quick washing in PBST, the membranes were blocked in 5% non-fat dry milk in PBS for at least 1 h and then incubated with primary antibodies (anti-MUC16 and Jak2). The immunoblots were washed five times (5 × 10 min), incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies, washed five times (5 × 10 min), reacted with enhanced chemiluminescence ECL reagent (Amersham Biosciences) and exposed to X-ray film to detect the signal.
Confocal immunofluorescence microscopy
MDA MB 231 cells with MUC16 knockdown and the control vector were grown on sterilized cover slips for 30 h. Cells were washed with Hanks buffer containing 0.1M HEPES, fixed in ice-cold methanol at −20 °C for 2 min and blocked with 10% goat serum (Jackson Immunoresearch Labs, Inc., West Grove, PA, USA) for at least 30 min. After the blocking step and a quick wash in PBS, cells were incubated with antibodies for MUC16, JAK2, Cyclin E, Cyclin A, p21, Cyclin B1 and phospho-Aurora kinase A for 60 min at room temperature. Then, cells were washed (4 × 5 min each washing) with PBS and incubated with fluorescein isothiocyanate-conjugated anti-mouse and Texas red-conjugated anti-rabbit secondary anti-bodies (Jackson Immunoresearch labs, Inc.) for 30 min at room temperature in the dark. 4′, 6-diamidino-2-phenylindole was used for nuclear staining. Cells were washed again (5 × 5 min) and mounted on glass slides in anti-fade Vecta-shield mounting medium (Vector Laboratories, Burlingame, CA, USA). Laser confocal microscopy was performed using an LSM 510 microscope (Carl Zeiss GmbH, Jena, Germany).
Synchronization and cell cycle analysis
MDA MB 231 and HBL 100 cells were grown in 100mm plates, and thymidine (Sigma) was added to the culture medium at a final concentration of 2mM for 12 h. Following two washes with serum-free medium, the cells were released from the thymidine block by culturing in fresh medium containing 24mM 2 deoxycytidine. After 9 h of incubation, the second thymidine block was initiated and completed after 14 h. The cells were released from the block by washing in warm phosphate buffered saline and replacing with complete culture medium. At different time points, the cells were fixed in 70% ethanol. After fixation, the cells were left on ice (~45 min) and then centrifuged. The pellets were resuspended in Telford’s reagent (90mM EDTA, 2.5mU of RNase A/ml, 50mg of propidium iodide/ml and 0.1% Triton X-100 in PBS). After incubating in an ice bath for ~2 h, the total DNA content was analyzed using the fluorescence-activated cell sorting method.
A total of 2 × 106 cells were seeded in 60mm petri dishes and allowed to grow for 48 h. The cells were then trypsinized and washed with PBS twice. Apoptosis was measured using the annexin V-fluorescein isothiocyanate apoptosis detection kit (Roche Diagnostics, Indianapolis, IN, USA). Apoptosis was detected by staining the cells with annexin V and propidium iodide solution followed by flow cytometry.
Inhibition of JNK1/2 in breast cancer cell MDA MB 231
MUC16-knockdown MDA MB 231 cells (2 × 106) were seeded, and after 12 h treated with JNK inhibitor. The JNK1/2 inhibitor SP600125 (20, 40 and 80 nM) was used to treat MUC16-knockdown MDA MB 231 cells for 12 h. After the inhibition of JNK1/2, protein was extracted from the cells for further western blot analysis.