Cell culture and treatments.
The murine C2C12 and C2C7 skeletal muscle cell lines were cultured in growth medium (GM; Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine). Normal Human Skeletal Muscle Myoblasts (HSMBs) was purchased from Clonetics® and cultured in Basal Medium with SingleQuots® as described in the data sheet (Lonza). Muscle differentiation was induced, exposing cells to differentiation medium (DM; Dulbecco's modifed Eagle's medium supplemented with 2% horse serum).
Single muscle fibers with associated satellite cells were isolated as described in reference 36
. Briefly, the hind limb muscles were digested with collagenase, and single myofibers were plated on matrigel (Sigma, 1 mg/ml ECM gel) coated dishes in GM1 (DMEM supplemented with 10% horse serum (Gibco), 0.5% chick embryo extract (MP biomedicals) and penicillin-streptomycin (Gibco) at 37°C). Three days later the medium was replaced with proliferation medium (GM2-20% FBS, 10% horse serum, 1% chick embryo extract in DMEM) to promote proliferation of detached cells (delaminated satellite cells). After 4–5 d, the cells were allowed to differentiate, replacing the medium with differentiation medium (DM-2% HS and 0.5% chick embryo extract in DMEM).
Genotoxic treatments were carried by incubating cells in GM for 12–16 h to the following DNA damaging agents: 0.4 µM Doxorubicin, 0.5 µM Etoposide, 75 µM MMS; when indicated, cells were pretreated 30 min with 5 mM caffeine or 1 µg/ml IGF-1 before drug exposure. The acute treatments were carried for 1 h with the following dosage: 3 µM Doxorubicin, 10 µM Etoposide, 250 µM MMS. After drug exposure, cells were incubated in DM for 24 up to 72 h.
Alkaline comet analysis.
C2C12 cells were treated with 250 µM MMS, 10 µM Etoposide and 3 µM Doxorubicin for 1 h then shifted in differentiation medium and harvested at different time points. DNA breaks and repair kinetics were measured as previously described in reference 37
with minor modifications. Single cells were analyzed with “TriTek CometScore version 1.5” software. The tail moment was used as measure of DNA damage. One hundred cells for each experimental point were scored.
Isolation of total RNA and microarray analysis.
HSMBs were cultured in Basal Medium with SingleQuots,® as described in the data sheet (Lonza), and then treated for 18 h with Doxorubicin 0.4 µM prior exposure to differentiation medium (DM) for 24 h. Total RNA was extracted with Trizol,® as described in the data sheet (Invitrogen).
RNA (500 ng) was reverse-transcribed by M-MLV reverse transcriptase. The transcripts were labeled with biotin using an RNA amplification kit (Ambion). The cDNA samples were mixed with a Hyb E1 hybridization buffer containing 37.5% (w/w) formamide. The hybridization mix was dispensed on the Sentrix Human Ref-8 BeadChip (Illumina) containing 24,000 transcripts of the 22,000 genes represented in the consensus Reference Sequence (RefSeq) human genome database. Hybridization was performed for 18 h at 55°C. Array chips were then washed with an E1BC solution, then with 100% ethanol and, lastly, with the E1BC solution again. The chips were blocked with an E1 blocking buffer followed by staining with streptavidin-Cy3, washing with the E1BC solution and drying. Array chips were scanned using a BeadArray Reader (Illumina). The resulting images were analyzed using the BeadStudio image processing software (Illumina). The chip contained 30 to 40 beads with the attached oligonucleotide DNA corresponding to an individual gene of the RefSeq database. Differentially expressed transcripts resulting from the comparison between the Doxorubicin-treated sample and the control sample (≥ 2-fold) were divided into upregulated and downregulated. Both subsets of genes were then analyzed for the presence of overrepresented GO categories (Biological Process), GeneGo process Networks and GeneGo Pathway Maps using MetaCore™ software from GeneGo Inc. Gene expression of XPC DDB2 and p21 was validated by RT-PCR using the following primers.
- Human p21:
- FWD: TGT CAC TGT CTT GTA CCC TTG
- REV: GGC GTT TGG AGT GGT AGA A
- Human XPC:
- FWD: GTC TCT ACA GCC AAT TCC TCT G
- REV: CCT TTG CTG GTC TTT GGT TTG
- Human DDB2:
- FWD: GGC TGC AAG ACT TTA AAG GC
- REV: ACA TCC AGG CTA CAA AAC CAG.
Protein gel blot and immunofuorescence.
C2C12 mouse cell line were treated with 0.4 µM Doxorubicin and MMS 75 µM for 16 h; when indicated, cells were pretreated with 5 mM caffeine and IGF-1 before genotoxic agents exposure. After drug treatments, cells were shifted in DM for 48 h. Proteins were extracted with Ripa buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Np40, 1 mM EDTA), separated on polyacrilamyde gel and transferred to nitrocellulose filters. The following primary antibodies were used to detect endogenous protein level: MF20 mouse monoclonal antibody to detect Myosin Heavy Chain (MyHC), monoclonal antibodies against Myogenin (F5D), MyoD antibody (M-318 rabbit polyclonal Santa Cruz), phospho-p53 Ser15 (Cell Signaling), Tubulin (Ab4 from NeoMarkers). Primary antibodies were visualized with the ECL (Amersham) chemioluminescent kit following the manufacture's instruction.
For immunostaining, HSMBs and satellite cells were plated on glass coverslips; when indicated, cells were treated with genotoxic agents as described above. After DNA damage exposure, cells were shifted in DM. After 4 days, cells were fixed with 3.7% formaldehyde and permeabilized 10 min with PBS supplemented with 0.2% Triton. Single or double fluorescence were performed with the following primary antibodies (Ab): MoAb MF20 for Myosin Heavy Chain (MyHC), rabbit polyclonal anti total NbsI (Novus), rabbit polyclonal anti phospho 139 H2AX (Upstate). We used rodamine-conjugated goat anti-mouse IgG and fluoresceine-conjugated goat anti-rabbit IgG secondary antibodies (Jackson Immunoreserch) to detect the primary Ab, according to manufacture's instructions. Nuclei were visualized by 4′,6′-diamino-2-phenylindole (DAPI).
RNA extraction and RT-PCR.
Total RNA was extracted with Trizol (Invitrogen) according manufacturer instructions. 0.5–1 µg of RNA was retrotranscribed using the Taqman reverse transcription kit (Applied Biosystems). Real-time quantitative PCR was performed to analyze relative gene expression levels using SYBR Green Master mix (Applied Biosystems) and following manufacturer indications. Primers sequences are as follows:
- Human mck:
- Fwd: GGC ACA ATG ACA ACA AGA GC
- Rev: GAA AAG AAG AGG ACC CTG CC
- Human myogenin:
- Fwd: GCC ACA GAT GCC ACT ACT TC
- Rev: CAA CTT CAG CAC AGG AGA CC
- Fwd: CAC CAT CTT CCA GGA GCG AG
- Rev: CCT TCT CCA TGG TGG TGA AGA C.
Chromatin inmunoprecipitation (ChIP).
ChIP assay on C2C12 was performed using the following antibodies: anti-acetylated histone 3 (Upstate), MyoD (Santa Cruz SC-760), H3-K4 tri-methylation (Millipore). Normal rabbit IgG (Santa Cruz, SC-2027) antibody was used as a control. Real-time PCR was performed on input samples and equivalent amounts of inmunoprecipitated material using the SYBR Green Master Mix (Applied Biosystems). Relative recruitment is calculated as the amount of amplified DNA normalized to input and relative to values obtained after normal rabbit IgG inmunoprecipitation, which were set as the background (one unit).
Primers used were as follows:
- Mouse myogenin promoter:
- Fwd: TGG CTA TAT TTA TCT CTG GGT TCA TG
- Rev: GCT CCC GCA GCC CCT
- Mouse mck enhancer:
- Fwd: AGG GAT GAG AGC AGC CAC TA
- Rev: CAG CCA CAT GTC TGG GTT AAT
- Human myogenin promoter:
- Fwd: GCC ATG CGG GAG AAA GAA G
- Rev: AGC CAA CGC CAC AGA AAC C
- Human mck enhancer:
- Fwd: CCT TGC CCT GAG TTT GAA TCT C
- Rev: GGC AGT CTA ACC CCA GAA ACC.
For cell cycle analysis, C2C7 skeletal muscle cells were treated 16 h with different DNA damaging agents and then shifted in DM for 24 h. Cells were collected and than stained for 30 min at 37°C with a solution containing propidium iodide at 100 mg/ml, RNase at 200 mg/ml and 0.2% Triton X-100 and analyzed with an EPICS XL cytofluorimeter (Coulter).