Mice and rats of either sex were housed at the Animal unit at CIC bioGUNE, and all procedures were approved by the institutional review committee on animal use. The CIC bioGUNE’s Animal Unit is an AAALAC accredited facility.
RNA immunoprecipitation (IP)
IP protocol of endogenous mRNA–transfected HuR complexes was performed as described in Keene et al. (2006)
. In brief, 500 μg of whole-cell lysate obtained from a pool of NB or P5 sciatic nerves from C57BL6J mice of either sex were incubated with a suspension of Protein A–Sepharose beads (Sigma-Aldrich), pre-coated with 15 μg of either IgG1 (BD Pharmingen) or anti-HuR (Santa Cruz Biotechnology) antibodies. For RIP-chip analysis, appropriate amounts of total RNA from four biological replicates of HuR and Mock IPs, as well as 2 replicates of input mRNA from NB and P5 nerves were submitted to the Genomics Analysis Platform at CIC bioGUNE for analysis on MOUSE WG-6 V2 arrays (Illumina). For RIP-qPCR analysis, bound mRNA was measured by real-time PCR analysis, normalized to GAPDH
mRNA bound in a non-specific manner to IgG1.
Microarray analysis and GO classification
Data were extracted using BeadStudio data analysis software. Data were processed and normalised using Robust Spline normalisation using Lumi bioconductor package (Du et al., 2008
). The data for the probes with a detection p-value higher or equal to 0.01 were excluded. Those detected with p-value lower than 0.01 in at least one array were accepted as significant. For the detection of differentially expressed genes, a linear model was fitted to the data and empirical Bayes moderated t-statistics were calculated using the limma package from Bioconductor. Adjustment of p-values was done by the determination of false discovery rates (FDR) using Benjami-Hochberg procedure (Smyth, 2005
; Peart et al., 2005
). Biological functional analysis was performed using the Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA). Fisher’s exact test was performed with the P
value threshold of 0.05 to identify molecular functional categories with statistical significance.
Biotin Pull-Down Assay
Biotinylated transcripts were synthesized using cDNA that was prepared from sciatic nerves. Templates were prepared using forward primers that contained the T7 RNA polymerase promoter sequence (CCAAGCTTCTAATACGACTCACTATAGGAGA), as described (Li et al., 2002
). Biotin pull-down assays were performed as described elsewhere (Antic and Keene, 1997
) and bound proteins were analyzed by Western blotting. In brief, the PCR-amplified fragments were purified and used as templates for in vitro
synthesis of the corresponding biotinylated RNAs by MAXIscript kit (Applied Biosystems). Biotin pull-down assays were performed by incubating 40 μg of P5 sciatic nerve cell lysates with equimolar amounts of biotinylated transcripts for 1 hr at room temperature. The complexes were isolated using paramagnetic streptavidin-conjugated Dynabeads (Invitrogen), and bound proteins in the pull-down material were analyzed by Western blotting using an antibody recognizing HuR. Primer sequences are available on request.
RNA isolation and quantitative PCR (qPCR)
RNA was isolated with Trizol (Invitrogen) and its concentration and integrity determined. qPCRs were performed using BioRad iCycler thermocycler. Ct values were normalized to the housekeeping expression (GAPDH). Primer sequences are available on request.
Protein isolation and Western blotting
Isolation and Western blotting of total proteins from cells and nerves were done as described (Parkinson et al., 2008
). Subcellular fractions were isolated using the Proteoextract Subcellular Proteome Extraction kit (Calbiochem), according to manufacturer’s instructions. The purity of cytoplasmic and nuclear fractions was examined by Western blotting in each experiment using antibodies to Gapdh and Histone H3 respectively.
Rat primary Schwann cell culture
Sciatic nerves obtained from P3 Wistar rats of either sex were digested in 0.25% trypsin, 0.4% collagenase in DMEM, and cultured for 3 days in DMEM with 10% fetal bovine serum (FBS) containing AraC (10−3 M). After 3 days in culture, Schwann cells were immunopanned to remove remaining fibroblasts and expanded in DMEM/F12, 3% FBS, NRG1 (10 ng/ml), N2 supplement and Forskolin (4 μM). Only the first 5 passages were used. For proliferation experiments, cells were first cultured overnight in Minimal Medium (MM: DMEM/F12, 5% FBS, and N2 supplement) before NRG1 (20 ng/ml) or TGFβ (2 ng/ml) stimulation at appropriate time-points. For cell shape experiments, cells were cultured overnight in MM before replating onto PDL- or laminin-coated dishes. For apoptosis assays, freshly isolated Schwann cells from NB nerves were isolated, plated onto coverslips in Simple Medium [SM: DMEM/F12 and BSA (0.3 mg/ml final)] and treated with TGFβ (2 ng/ml) for 48 hrs. Recombinant human TGFβ1 and NRG-1 (Heregulin-β1 isoform) were purchased from Peprotech and R&D Systems respectively.
Neuron-Schwann cell co-cultures
Myelinating neuron-Schwann cell co-cultures were prepared by adding purified rat Schwann cells, infected with sh HuR lentivirus or adenovirus expressing HuR, to purified E15 rat DRG neurons. Myelination was induced with 50mg/ml ascorbic acid and MBP antibodies were used to label myelin segments or RNA extracted (Parkinson et al., 2008
For HuR knockdown, cells were treated with short-hairpin lentiviral particles against HuR [CCGGCCCACAAATGTTAGACCAATTCTCGAGAATTGGTCTAACATTTGTGGGTTTTTG] in the presence of hexadimethrine bromide (8 μg/ml). After 24 hrs transduction, the cells were selected using puromycin (1.25 μg/ml) and puromycin-resistant HuR-knockdown cell clones were grown, analyzed, and frozen for future use. For adenoviral infections, cells were cultured in MM, adenoviral particles added (amount added determined by titration) and 24 hrs later the medium changed. Adenoviral constructs used were: GFP/Krox-20 (Ad-K20) and its matched GFP control (Ad-GFP), a gift from J. Milbrandt (Parkinson et al., 2004
); SMAD7 (Ad-SMAD7) (Blaney-Davidson et al., 2006
) and HuR (Ad-HuR) (Xiao et al., 2007
Specific inhibitors in our culture experiments were obtained from Calbiochem and were used at the following concentrations: 10 μM UO126 (Erk 1/2 inhibitor), 10 μM SB203580 (p38 inhibitor), 10 μM LY294002 (PI3K inhibitor), 10 μM MG132 (Proteosome inhibitor) and 2 μM BAY11-7082 (NF κB inhibitor).
Migration using the “scratch-assay” was performed as described previously (Liang et al., 2007
). In brief, control and HuR
-silenced cells were seeded onto PDL- or laminin-coated dishes and cultured overnight in MM. A scratch was performed using a p200 pipette tip, cultured medium changed and pictures taken at time 0, 6, 12, 18 and 24 hrs. The gap distance was measured at each time-point and data expressed as percentage gap distance over time.
cAMP myelination assay
A cAMP analogue, dibutryl cAMP (Sigma, UK) was added to cultures (10−3M), and protein or mRNA obtained 24 hrs later.
Immunohistochemistry (IHC) and immunocytochemistry (ICC)
For teased nerves, nerves were dissected out, immediately fixed in 4% PFA for 10 min, teased on microscope slides and allowed to dry. The samples were incubated in 0.2% triton in blocking solution (BS: PBS containing 10% calf serum, 0.1% lysine and 0.02 % sodium azide) followed by overnight incubation at 40
C with the following primary antibodies: HuR (1:100) and TUJ1 (1:5000) followed by secondary antibodies conjugated with FITC or Cy3 (Cappel/Jackson Immunoresearch Labs). Talin ICC has been described elsewhere (Nodari et al., 2007
), and in vitro
BrdU incorporation and Tunel labeling described elsewhere (D’ Antonio et al., 2006a
; Parkinson et al., 2001
). Images were acquired with an AxioImager D1 fluorescent microscope (Zeiss).
Chromatin Immunoprecipitation (ChIP)
Rat Schwann cells or whole nerves were cross-linked with 1% formaldehyde (vol/vol) at 25 °C for 10 min. After sonication into 200–500-bp fragments using Bioruptor (Diagenode), chromatin was immunoprecipitated with 2 μg of anti-p65 antibody (sc-372, Santa Cruz Biotechnology) or 5 μg of anti-Smad2/3 antibody (3102, Cell Signaling Technology) using the Magna ChIP G kit (Millipore). The recovered DNA was subjected to PCR amplification. Chromatin that was immunoprecipitated with mouse IgG was used as a negative control. The abundance of target genome DNA was calculated as the percentage of input. Primer sequences are available on request.
Antibodies used were from: HuR and p65 (Santa Cruz Biotechnology), Gapdh (abcam), β-actin and Talin (Sigma), p-ERK1/2, p-AKT, p-p38, Smad 2/3 (Cell Signalling Technology), Egr2 and TuJ1 (Covance), MPZ (Astexx), Periaxin (gift from P. Brophy), MBP (Eurogentec). Fluorescent-conjugated secondary antibodies were from Jackson Immunoresearch and HRP-conjugated secondary antibodies were from Biorad.
Assessment of mRNA stability
mRNA stability was determined by actinomycin D chase experiments, following a standard protocol described elsewhere (Chang et al., 2010
). Briefly, control and HuR
-silenced cells were treated with db cAMP for 24 hrs, which significantly increased expression of all mRNAs analysed. Actinomycin D was added to a final concentration of 5 μg/ml to block further transcription. At 0, 30, 60, 120 and 240 min after actinomycin D treatment, the cells were harvested and mRNA was quantified by qPCR. The mRNA decay was recorded as the percentage of mRNA remaining over time compared with the amount before the addition of actinomycin D.
All data are presented as arithmetic mean ± standard error of the mean (SEM) unless otherwise stated. Statistical significance was estimated by the Student’s t-test.