To create expression vectors encoding C-terminally V5-tagged fusion proteins, cDNA sequences were PCR amplified and cloned into pcDNA 3.1 D/V5-His-TOPO (Invitrogen, Carlsbad, CA). NET9, NET32, and NET39 DNA sequences were PCR amplified from the cDNA clones ATCC 10159451, MGC-27952 and MGC-28994 respectively. The primers for PCR amplification of these were
NET9 Fwd: CACCATGTCCCAAAGTCTGAAGAGCCA,
NET9 Rev: CAAAAGGCAGCCTCGCTCTTC,
NET32 Fwd: CACCATGGCTCAGTTGGGAGCTGTTG,
NET32 Rev: GTTCTCCTTTGTGGGTGCCTC,
NET39 Fwd: CACCATGCCAGCTTCCCAGAGCCG, and
NET39 Rev: CCAGGCAGAGATGAGCATCTG. NET14a, NET25, NET37 and calnexin were PCR-amplified from bulk cDNA that we prepared from RNA extracted from C2C12 cells using Transcriptor First Strand cDNA Synthesis Kit™ (Roche Diagnostics, Alameda, CA). The primers for PCR amplification were
NET14a Fwd: CACCATGGCTACAGAAATTGGCTCT,
NET14a Rev: CACAGTAGATAATAAAAAGGAATAACG,
NET25 Fwd: CACCATGGCCGGCCTGTCGGAC,
NET25 Rev: TCGCTCTGAATCAGAGAAAGAGGA,
NET37 Fwd: CACCATGTCCCAGAACCTTCAGGAG,
NET37 Rev: GGACGCCCAGGTAAAGTAGGC,
Cnx Fwd: CACCATGGAAGGGAAGTGGTTACTG, and
Cnx Rev: CTCTCTTCGTGGCTTTCTGTT. NET14b cDNA (WDC146-EGFP) was a kind gift of Dr. Akiko Sakai (Okayama Univ., Okayama, Japan). Rat LAP2β cDNA was previously described [22
Anti-peptide antibodies to NETs were prepared by immunization of rabbits with synthetic peptides coupled to pre-activated keyhole limpet hemocyanin (KLH, Imject™, Pierce Biotechnology, Rockford, IL). Each rabbit was injected with a mixture of 2–3 different peptides derived from the same NET, comprising the following sequences (with each peptide containing an N-or C-terminally added cysteine for conjugation to KLH): NET25, residues 49–59 and 63–81; NET32, residues 262–277, 311–325 and 326–340; and NET37, residues 1–15, 18–32 and 322–336. For affinity purification, each peptide was conjugated to CNBr-activated Separose at a concentration of 0.5 mg peptide/ml beads. Typically, antibodies were affinity-purified using a column containing a mixture of the 2–3 peptides used to immunize the animal. Antibodies were eluted from the affinity columns with 4 M MgCl2, and were dialyzed against PBS. Polyclonal antibodies to lamins A/C and lamin B1 were prepared in rabbits using the pertinent recombinant proteins, and affinity purified using columns coupled with recombinant proteins. Antibodies to the following antigens were obtained from commercial sources: V5 epitope tag (mouse monoclonal, Invitrogen); desmin and emerin (mouse monoclonal, Novocastra, UK).
C2C12 myoblasts (ATCC # CRL-1772) were grown in Dulbecco's MEM supplemented with 20% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. To induce myoblast differentiation [57
], 5 × 105
C2C12 cells were seeded into a 10 cm, gelatin-coated petri dish. 16 h later, cells were shifted into "low serum" medium consisting of Dulbecco's MEM supplemented with 2% horse serum and antibiotics. This time point was termed "day 0". Multi-nucleated myotubes could be seen by day 3, and increased in number and size by days 5–6. Typically, at least 50% of the nuclei were in multinucleated cells by day 6, the standard time point used for the differentiation experiments.
Transient transfection of C2C12 cells
C2C12 cells were seeded in 6-well microtiter plates containing gelatin-coated glass coverslips at 1 × 105 cells/well and allowed to grow for 16 h. Prior to transfection, the growth media containing 20% FCS was removed from cells and replaced with Opti-MEM™ (Invitrogen). cDNA expression plasmids (1μg/well) were transiently transfected into cells using Lipofectamine 2000™ (Invitrogen) at 1:3 ratio (DNA [μg] vs. Lipofectamine [μl]) according to the manufacturer's instructions. The transfection mix was removed from cells 6 hours later and either replaced with the high-serum growth medium to maintain cell proliferation, or with the low-serum media to induce cell differentiation. Proliferating cell cultures were maintained for another 18 to 36 h, whereas differentiated cells cultures were maintained to day 6, and immunofluorescence localization was carried out (below paragraph).
Cells were fixed with 4% paraformaldehyde in PBS for 5 minutes at RT, either with or without pre-extraction with Triton X-100 before fixation. For Triton pre-extraction, cells were rinsed once with PBS and then extracted with 1% Triton X-100 in 10 mM Tris-HCl pH 7.5, 1.5 mM MgCl2 and 2 mM CaCl2 for 30 seconds at RT. Following fixation, cells were treated with 0.5% Triton X-100 in PBS and then incubated for 1 h with a mouse monoclonal antibody against the V5-epitope tag, followed by washing in PBS and incubation for 1h with FITC-conjugated goat-anti-mouse secondary antibody. Chromosomal DNA was stained with TOPRO-3 (Invitrogen, Carlsbad, CA). The stained cells on cover slips were subsequently mounted in Gel Mount™ (Biomeda, Foster City, CA) and sealed with Clarion™ (Biomeda). Cell images were taken with the Bio-Rad/Zeiss MRC1024 laser scanning confocal microscope and analyzed by LaserSharp 2000™ software (Bio-Rad). Images were pseudo-colorized and merged with Adobe Photoshop CS™.
To extract RNA from C2C12 cells, proliferating or differentiated cultures grown on 10-cm petri dishes were rinsed once with PBS and directly lysed in 1 ml of Trizol™ (Invitrogen) following the manufacturer's instructions. RNA samples were further treated with TURBO DNase™ (Ambion, Inc., Austin, TX) and purified by RNeasy Total RNA Isolation Kit™ (Qiagen, Valencia, CA). RNA purity was tested by spectrophotometry, and RNA integrity was verified by agarose gel electrophoresis to visualize ribosomal RNA. To limit variance in the microarray experiments, each RNA sample that was analyzed was pooled from 3 separate culture plates that were grown at the same time, and 3 such RNA pools prepared from cultures set up at sequential one-day intervals were further combined. To extract RNA from mouse tissues, the 10 tissues or organs indicated were surgically removed from C129 mice on embryonic day 17 (E17), post-natal day 7 (P7) or ~10 week old mice (adult). Care was taken to remove fat from hindlimb skeletal muscle. Upon removal, tissues were immediately immersed in RNA Later™ (Ambion, Inc., Austin, TX) and stored at -80°C to preserve RNA integrity. Tissues or organs from 3–4 mice were pooled and homogenized in a hand-held homogenizer. RNA samples were extracted and purified as described above.
DNA microarray analysis
DNA microarray hybridization was carried out by the TSRI DNA microarray core facility [58
]. Briefly, 5 μg of total RNA were used to synthesize cDNA that was then used as a template to generate biotinylated cRNA. cRNA was fragmented and hybridized to the Affymetrix GeneChip Mouse Genome 430 2.0™ array (Affymetrix, Santa Clara, CA), which allows analysis of over 39,000 transcripts selected from GenBank, dbEST, and RefSeq. The probe set for each gene on this microarray chip contains 11 perfectly matched probes and 11 control probes with a 1 bp-mismatch; moreover, some genes are represented by multiple probe sets (Additional File 1
). The arrays were then washed and scanned with a laser scanner, and images were analyzed by using Affymetrix Microarray SuiteVersion 5.0™ (MAS 5.0; Affymetrix). The raw data of microarray signal values were deposited in Gene Expression Omnibus (GEO accession #GSE4694). Three different RNA samples (see above) from proliferating and from differentiated C2C12 cells were individually analyzed (6 microarrays total). The data were further normalized by GCRMA [59
] to eliminate probe level anomaly, and were subjected to a Bayesian regularized t
], comparing proliferating cells ("baseline") and differentiated cells ("experimental"), to obtain a global false discovery rate measurement and confidence level assessment, which is indicated as a p
-value for the significance of expression changes for each probe set. A list of probe sets used to measure NET expression level is provided in Additional File 1
. The expression change was calculated by dividing the experimental (differentiated cells) signal values by the baseline (proliferating cells) values for up-regulated genes, and vice versa
for down-regulated genes. Only data from probe sets with a p
-value of less than 0.05 in the regularized t
-test were deemed "significant" and were used to calculate the average expression change of NETs.
Quantitative real-time PCR
Quantitative real-time PCR was performed using a LightCycler 2.0 thermal cycler system according to the manufacturer's instructions (Roche Diagnostics). RT-PCR primers were designed to target the sequences close to Affymetrix probe regions and the sequences across the exon-exon junction when it was possible (Additional File 1
). 2 μg of total RNA was first reverse-transcribed to cDNA using Transcriptor First Strand cDNA Synthesis Kit™ (Roche Diagnostics). The cDNA sample was then used to measure expression level of a gene. The targeted gene was amplified using LightCycler FastStart DNA MasterPLUS
SYBR Green I™ (Roche Diagnostics) with a Touch-Down PCR protocol, where annealing temperature was ramped down from 68°C to 55°C to target a broad range of probe annealing temperatures. A standard curve that was used to correct different amplification efficiencies was generated for each gene using a dilution series of cDNA prepared from C2C12 total RNA. Two housekeeping genes, Hprt and Ppia, were chosen for "Reference" transcripts in every quantitative PCR run because both genes had consistent expression levels across all tested samples. The "Calibrator", a cDNA sample prepared from C2C12 total RNA in bulk, was also included in each PCR run to correct variances among reagents and thermocycler runs.
To quantify levels of NE proteins and desmin at day 0 and day 6 after differentiation, 3 different C2C12 differentiation experiments conducted on separate days were examined. Total cell protein was harvested on day 0 and day 6 after induction of differentiation, and equal amounts of total protein from each sample were loaded on SDS gels. Proteins were separated by electrophoresis, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis. The analysis was performed using primary antibodies against lamins A and C, lamin B1, NET25, emerin, histone H2B and desmin, and horseradish peroxidase-conjugated secondary antibodies. Proteins were detected by SuperSignal West Pico™ (Pierce). X-ray films were scanned on Bio-Rad GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA). The intensities of the corresponding protein bands were quantified by QuantityOne software (Bio-Rad). Similar methods were used to compare the concentrations of NETs in isolated NEs and MMs.
Subcellular fractionation of mouse liver
Mouse liver NEs were isolated as described [61
], and mouse liver MMs were prepared as before [22
]. The analysis in Fig. involved NEs that were pre-extracted with 0.5 M NaCl to remove most chromatin contamination [61
Colorimetric determination of phospholipids
Phospholipids in mouse liver NE and microsomal membrane (MM) fractions were determined using a miniaturized method based on the complex formation between phospholipids and ammonium thiocyanate [62
]. Briefly, 20 μl of NEs or microsomal membranes (MMs) were extracted with 400 μl chloroform: methanol (2 : 1) and concentrated. Samples were diluted with chloroform to a total volume of 150 μl and mixed with an equal volume of ammonium thiocyanate. The amount of water insoluble complex was determined spectrophotometrically at a wavelength of 460 nm. Phospholipid concentrations were determined using a standard curve produced with L-α-phosphatidylcholine dissolved in chloroform (Sigma, St. Louis, MO).