Antibodies against α1, α2, α5 and β1-integrin subunits for flow cytometric analysis were a kind gift from B. Chan (Universtiy of Western Ontario, Canada). Biotinylated β3-integrin antibody was purchased from Pharmingen (Bedford, UK). αv-integrin antibody was purchased from Chemicon International (Harrow, UK). α6-integrin antibody was purchased from ABD Serotec (MorphoSys Ltd, Kidlington, UK). β4-integrin antibody was purchased from AbCam (Cambridge, UK). HRP and FITC-conjugated anti-rabbit, anti-rat and anti-mouse antibodies were all purchased from Jackson Immuno Research Labs (Baltimore Pike, PA, USA) or from Biosource (Nivelles, Belgium). Myosin VI and myosin VIIa were purchased from Abcam (Cambridge, UK). For Western blot analysis, αv and β1-integrin antibodies were purchased from Chemicon International. β3-integrin and HSC-70 antibodies were obtained from Santa Cruz (Wiltshire, UK).
OC-2 cell culture
OC-2 cells (gift from Prof M. Holley, Sheffield University, UK)  were cultured at 33°C in a 5% CO2 atmosphere in DMEM + 10% FCS + 10% glutamine in order to express the immortalisation gene, small tumour antigen (Tag). Under these conditions the cells can be passaged multiple times by detaching the cells from the flasks with trypsin/EDTA (1x T3924 Sigma). To induce differentiation, the cells were cultured at 39°C in 5% CO2 in DMEM + 10% FCS + 50 U/mL-1 IFNγ + 10% glutamine for 14 days to inactivate the immortalisation gene. The phenotype of undifferentiated and differentiated OC-2 cells was confirmed by examining the cellular morphology and expression of myosin VI and myosin VIIa expression levels, prior to use in experiments.
Flow cytometry analysis
Single cell suspensions of undifferentiated and differentiated OC-2 cells were incubated with either anti-α1, -α2, -α5, -α6, -αv, -β1, -β3 or -β4 antibodies followed by incubation with a FITC-conjugated secondary antibody. Cells were then washed and resuspended in FACS buffer (0.1% BSA in PBS) and analysed for fluorescence using a Becton Dickinson FACS Calibur flow cytometer. For negative controls, IgG matched isotype controls were used. Flow cytometry experiments were repeated 3-9 times.
For retroviral transduction experiments, transduced cells were screened by FACS analysis using a human β3-integrin antibody, LM609 (BD Pharmingen), to determine the level and efficiency of transduction.
OC-2 retroviral infection
Undifferentiated OC-2 cells were grown to 60% confluency and transduced with retrovirus expressing either the human β3-integrin (gift from J. Marshall, QMUL, London) or an empty expression cassette, for 48 h at 33°C. After 48 h incubation, the viral medium was removed and replaced with OC-2 cell culture medium followed by further incubation for 48 h. OC-2 cells infected with β3-integrin were selected from the non-infected cells by magnetic bead sorting (Dynal, Invitrogen, UK) using the antibody against β3-integrin (LM609). Cells were either lysed for Western blot analysis of myosin VI and myosin VIIa or trypsinised for FACS analysis of mouse and human β3-integrin surface levels.
Western blot analysis
Undifferentiated and differentiated OC-2 cells were grown to 70-80% confluency and lysed using RIPA buffer. Equal amounts of protein from each cell type were analysed by SDS-PAGE followed by transfer to nitrocellulose membranes (Hybond-ECL, Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked for 1 h in either 5% milk-PBS-Tween (0.05%) followed by incubation with antibodies against αv-, α6-, β1- and β3-integrin or myosin VI and myosin VIIa, overnight at 4°C. Membranes were washed 3 times with PBS-Tween (0.05%) followed by incubation with an HRP-conjugated secondary antibody for 1 h at room temperature. Chemiluminescence was detected by exposure to high performance chemiluminescence film (Amersham Biosciences, UK). Densitometric readings were obtained using Lab Images software. HSC-70 was used for a loading control.
Adult mice (Mus-musculus CBA/Ca) were killed by cervical dislocation according to Home Office regulations. Total RNA was isolated both from mouse cochlear tissue and from differentiated and undifferentiated OC-2 cells using the RNeasy mini kit from QIAGEN (Bio-Rad, Hercules, CA, USA). RNA was reverse transcribed using the Superscript III First-strand Synthesis kit (Invitrogen). The primers used to identify the different integrins were as follows (F = Forward, R = Reverse): α6 F: AGGAGTCGCGGGATATCTTT; α6 R 5'- GTAACAAAGCTCAGGGCTGC; αv F: CCAGCCCATTGAGTTTGATT αv:R: AAATCTTCAATGCCGTCACC; β1 F: GCTCCTCTTCCCCTCCATAC; β1 R GTAACAAAGCTCAGGGCTGC; β3 F GCTACAAACACGTGCTGACG; β3: R GAGGCAGAGTAGTGGTTGTCG β4 F GATGCCTACCCAGTCCTCAA β4 R TTCATAGGGCCACTCCAGAC.
The reaction conditions of the above sequences were as follow: denaturation at 94°C for 2 min; extension at 58°C for 45 s; and annealing at 72°C for 60 s for thirty cycles. PCR products were separated on a 1% agarose gel by electrophoresis for 1 h at 100 V.
Integrin siRNA transfection
One day prior to transfection, differentiated OC-2 cells (39°C) were counted and seeded at 1 × 105 cells per T-25 flask in medium without antibiotics to generate a 50-60% confluent monolayer on the day of transfection. A pool of four different oligonucleotides (siRNA SmartPool, Dharmacon, USA) was used to knockdown each single integrin mRNA efficiently. The integrin oligonucleotide sequences were as follows (5'-3'):α6: UUAACCUUGAGGCAUAUCCUU; UUGUUCUACACGGACGAUCUU; UUAAUGUAGACGUAAACUGUU; UGAUCCACCAAGCUACUCCUU. αv: UACUCAACGGUCUUUGUGCUU; AAAUGCUAGGGUACACUUCUU; UUCACGUACAGGAUUGCGCUU;UCGAUUGGCAGGUUCUUGUUU. β1: UCAUUCAUCAAAUCCGUUCUU; UUAAUGUAAACUUCUGUGGUU; UGAAACUUGGGAUCUGUGCU; GUAAUCUUCAGCCCUCUUGUU. β3: UAUACAGCGGGUUGUUUGCUU; UUCUCCUUCAGGUUACAUCUU; GUAGUAGCCAGUCCAGUCCUU; UAGUUUCUCAGUCAUCAGCUU
For one T-25 flask of cells, 2.5 μl of a 40 μM stock oligonucleotide solution was mixed, in a tube, with 182.5 μl of OPTIMEM to give a final volume of 185 μl. Oligofectamine, 3 μl (Invitrogen, Paisley, UK) was diluted in OPTIMEM to give a final volume of 15 μl and incubated for 5-10 min at room temperature (RT). The two solutions were then combined together to give a final volume of 200 μl and allowed to form a complex by incubating for 15-20 min at RT. The solution was added to each T-25 flask containing the cells to be transfected in 800 ul serum free medium. Cells were returned to the incubator set at 37°C in 8% CO2. Four hours after transfection, 500 μl of cell culture medium containing 3X the normal concentration of serum without antibiotics was added to each flask. Gene knockdown was assessed by FACS analysis 24 h after transfection. After verification of positive knockdown by FACS analysis, protein and RNA extraction was performed. Transfection with a scrambled oligonucleotide was carried out as control and shown to have no change on integrin expression
Statistical significance was calculated using Student's t-test. P < 0.05 was considered statistically significant.