Experimental Animals
Lgr5-EGFP-Ires-CreERT2(Lgr5-EGFP) mice33(Jackson Laboratory,Stock No.00887) and wide-type mice were raised in a comfortable environment with suitable temperature and light and fed with standard laboratory food and water ad libitum. We are approved by the Animal Care and Use Committee of Southeast University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All the operations were carried out in accordance with the procedures.
RNA extraction and RT-PCR
About 20 wild-type mouse cochleae were dissected to extract total RNA, which was reverse transcribed into cDNA with the cDNA Synthesis Kit (Thermo Fisher Scientific, K1622). Gene expression was measured by RT-PCR with GAPDH as the endogenous reference gene. The primers were as follows: GAPDH: (F) 5’-AGG TCG GTG TGA ACG GAT TTG-3’; (R) 5’-TGT AGA CCA TGT AGT TGA GGT CA-3’; AFF1: (F) 5’-GAA GGA AAG ACG CAA CCA AGA-3’; (R) 5’-TAG CTC ATC GCC TTT TGC AGT-3’; AFF4: (F) 5’-ATG AAC CGT GAA GAC CGG AAT-3’; (R) 5’-TGC TAG TGA CTT TGT ATG GCT CA-3’; ELL3: (F) 5’-GAC CAG CCT CCT GAT GCT AAG-3’; (R) 5’-GCC ACC ATT AGT GCC CTC TTG-3’.
Western blotting
About 10 cochleae from postnatal day (P)3 mice were dissected in order to extract proteins. GAPDH was used as the reference protein. The primary antibodies were anti-AFF1 (Sigma-Aldrich, #SAB2106246), anti-AFF4 (Santa Cruz, #sc135337), and anti-ELL3 (Abcam, #ab67415). Peroxidase-conjugated goat anti-rabbit (Life, A-31572) and goat anti-mouse (Invitrogen, A21202) were used as the secondary antibodies.
Cell Culture
HEI-OC1 cells were cultured in DMEM with 10% fetal bovine serum and 1% ampicillin at 37°C and 5% CO2. The cells were divided into two groups. The experimental group was treated with flavopiridol (AbMole M1710) at the concentration of 10 µM. Control cells were treated with DMSO in the same culture medium. After 12-hour culture, cells were treated with 0.25% trypsin/EDTA and then ultrasonicated (BioruptorTM UCD-200) for CDK9 kinase detection.
CDK9 kinase assay
Isolation of Lgr5+ progenitors via flow cytometry
About 50–60 cochleae were isolated from P0–P3 Lgr5-EGFP mice and then treated with 0.125% trypsin/EDTA (Invitrogen, 25200114) at 37°C. Trypsin inhibitor (10 mg/ml, Worthington Biochem) was added after 10 minutes to terminate the reaction. The trypsinized cochleae were pipetted up and down 80–100 times to obtain single cells, and the cells were then filtered through a 40 μm cell strainer (BD Biosciences, 352340). Dissociated cells were sorted on a flow cell sorter (BD FACS Aria III). The EGFP+ cells were collected as Lgr5+ progenitors for further in vitro cell culture experiments.
Sphere-forming assay and differentiation assay
Sorted Lgr5+ cells were cultured in DMEM/F12 medium at a density of 2 cells/μl (200 cells per well) for 5 days for sphere forming. The formula of DMEM/F12 medium was the same in previous study28. Spheres were identified with the Live Cell Imaging System and quantified using Image J. For differentiation, cells were cultured in the DMEM/F12 medium described above at a density of 20 cells/μl (2,000 cells per well) for 10 days. EdU (10 µM, (Invitrogen, C10420)) was added to label proliferating cells from day 4 to day 7. Flavopiridol (AbMole, M1710) was added to the experimental group from day 1 to day 10 at a concentration of 10 µM, while DMSO was added to the control group. Differentiated neurospheres were analyzed by immunofluorescent staining.
Immunofluorescent staining
The cochleae were dissected in cold HBSS in order to prevent protein degradation and then fixed with 4% PFA for 1 h at room temperature. In vitro cultured neurospheres were also fixed with 4% PFA for 1 h at room temperature. After washing with PBST three times, the cochleae or neurospheres were blocked with blocking solution for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies. The primary antibodies used were anti-Myosin7a (Myo7a; Proteus Bioscience, #25-6790; 1:1000 dilution), anti-Sox2 (1:400 dilution), anti-AFF1 (1:400 dilution), anti-AFF4 (1:50 dilution), and anti-ELL3 (1:400 dilution). After washing again three times, the cochleae or neurospheres were further incubated with secondary antibodies (Invitrogen, A21131, A21124) diluted 1:400 in PBT2 for 1 h at room temperature. After washing three times, the cochleae or neurospheres were mounted on slides with anti-fade fluorescence mounting medium (DAKO, S3023). Images were captured by Zeiss LSM 710 confocal microscope and analyzed by Image J software.
Statistical analysis
All the data in this research are presented as means ± SEM, and all experiments were repeated at least three times. All statistical analyses were performed in GraphPad Prism 5. P-values were calculated using a two-tailed, unpaired Student's t-test, and a p-value < 0.05 was considered statistically significant.