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Validation of the C. elegans model using juglone as a positive control for oxidative stress

ABSTRACT: To confirm the suitability of C. elegans as a model organism, preliminary gene expression experiments were performed using juglone (5-hydroxy-1,4-naphthoquinone; 97% pure; Sigma-Aldrich, St-Louis, MO), an ROS-generator, as a positive control of oxidative stress (d'Arcy Doherty et al. 1987; Leiers et al. 2003). Reproduction tests were initially performed to establish the sub-lethal effect concentrations used in the microarray experiments.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE59475 | GEO | 2014/07/17

SECONDARY ACCESSION(S): PRJNA255403

REPOSITORIES: GEO

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Project description:The annotation of the Affymetrix HTA 2.0 array was updated to optimise the detection of RNA in human skeletal muscle samples by removing invalid and low signal-high-variance probes Human primary skeletal muscle cells were isolated from muscle biopsies from healthy adults as previously described (Crossland et al. 2016; Crossland et al. 2017). Myogenic cell enrichment used magnetic-activated cell sorting (MACS) and anti-CD56 microbeads (130-050-401; Miltenyi Biotec). Myogenic purity was assessed through measurement of fractional desmin positivity (Crossland et al. 2016; Crossland et al. 2017). Cells were washed in PBS and fixed in ice-cold 1:1 acetone/methanol, before being blocked in 5% (v/v) goat serum for 30 min at room temperature. Cells were subsequently incubated with rabbit anti-desmin monoclonal antibody (ab32362; Abcam) for 1h at room temperature, washed, and incubated with anti-rabbit TRITC-conjugated secondary antibody as previously detailed (Crossland et al. 2016; Crossland et al. 2017). Finally, cells were washed in PBS and mounted/DAPI-stained using FluoroshieldTM mounting medium with DAPI. Myoblasts at passage 5-6 were cultured on Collagen Type I-coated 6-well dishes in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Life Technologies) containing 20% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), 1% (v/v) antibiotic-antimycotic (AbAm) solution and 4mM L-glutamine (Life Technologies). Cells were maintained at 37oC with 5% CO2. Once cells reached ~95% confluency, differentiation was induced by switching the medium to DMEM/F-12 containing 2% (v/v) horse serum (Sigma-Aldrich), 4mM L-glutamine and 1% (v/v) AbAm solution (Life Technologies). Six days following the initiation of differentiation, a media change was carried out, using differentiation media supplemented with long R3 IGF-1 (10 ng/ml; Sigma-Aldrich), with or without 100 nM rapamycin (Sigma-Aldrich). DMSO (final concentration 0.01% (v/v)) was used as a vehicle control for rapamycin. Samples were collected at 0h baseline and following 4h and 24h treatment.

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Validation of the C. elegans model using juglone as a positive control for oxidative stress

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