Oxyblots and Western blots
~20mg of muscle tissue was homogenized in 500 μL of RIPA lysis buffer containing 1% protease inhibitors cocktail. For cell experiments, C2C12 myotubes in 6-well plates were collected and homogenised in 100 μL of RIPA lysis buffer. The samples were then centrifuged for 10 min at 14000 x g rpm, 4°C. The supernatant was collected for immediate use or stored at -80°C. Protein concentrations were determined through use of commercially available colorimetric bicinchonnic acid (BCA) protein kit (Thermo Fisher Scientific, USA) to standardize the loading amount for the SDS-PAGE. SDS-PAGE was conducted as previously described (Chan et al., 2013) with specific antibodies against Phospho-eIF2α (Ser51, Cell Signaling Technology, USA #3398), Phospho-S6 Ribosomal Protein (Ser235/236, Cell Signaling Technology, USA #4858), Phospho 4E-BP1 (Thr37/46, Cell Signaling Technology, USA #2855), Fbx32/MAFbx (Abcam, USA ab168372), 19S proteasome (Abcam, USA ab20239), LC3A/B (Cell Signaling Technology, USA #12741), p62 (Cell Signaling Technology, USA #23214), actin (Cell Signaling Technology, USA #8457).
For oxyblots, the extracted protein samples were derivatised and stabilised using the OxyBlot Protein Oxidation Detection kit (Merck, Massachusetts, USA) for immunoblot detection of carbonyl groups, according to manufacturer’s instructions. For cell experiments, the protein samples were solubilised by boiling in 1x Laemmli sample buffer containing 10% 2-mercaptoethanol for 10 min. The samples were then loaded into 10% acrylamide gel for SDS-PAGE and immune-detection using the chemiluminescence method as previously described (Chan et al., 2013). Densitometry analysis was performed using the ImageLab software (Bio-Rad Laboratories).
Immunofluorescence and myotube diameter analyses
C2C12 myotubes grown on coverslips coated with Matrigel matrix basement membrane (Sigma-Aldrich, USA). At the end of experiment, the myotubes were fixed in 4% paraformaldehyde in 1X PBS at room temperature for 30 min and the excess was quenched with 300µM glycine. The fixed myotubes on coverslips were blocked and permeabilised in 10% bovine serum albumin, 2% Triton-X in 1X PBS for 1 hr at room temperature. The coverslips were then rinsed with 0.1% Tween 20 in 1X PBS and incubated overnight at 4°C with fluorophore conjugated antibodies against skeletal muscle myosin (F59 clone; AlexaFluor 488, Santa Cruz Biotechnology, USA). Unbound antibodies were removed by rinsing the coverslips in PBST, excessive moisture was removed before mounting in Fluoroshield with DAPI (Sigma-Aldrich, USA). Fluorescence images were captured using VS120 Olympus Virtual Slide Microscope (Olympus Life Science, Australia). The captured images were analysed using the Olympus cellSens software (Olympus Life Science, Australia). A minimum of 270 myotube diameters were measured for each condition.
Cell viability assay
The CellTiter 96 Aqueous One Solution (MTS) Cell Proliferation Assay (Promega, Australia) was used to determine cell viability of C2C12 myotubes, according to manufacturer’s instructions. Briefly, C2C12 myotubes stimulated with cigarette smoke extract (CSE) or hydrogen peroxide (H2O2) were washed and incubated in media containing MTS reagent at 37°C and 5% CO2 for 1 hour. Absorbance was then recorded at 490nm using a plate reader (CLARIOstar Monochrome Microplate Reader; BMG Labtech, Australia). To validate the specificity of the assay, MTS reagent was added to unseeded culture plate containing CSE or H2O2 for 1 hr before absorbance reading. No significant absorbance changes were observed in response to these stimuli verifying the reliability and specificity of the assay.
Enzyme-linked immunosorbent assay (ELISA)
Mature IL-6 and IGF-1 released by the C2C12 myotubes were quantified using commercially available ELISA kits: murine IL-6 ELISA Kit (Thermo Fisher, USA) and murine IGF-1 DuoSet ELISA Kit (R&D Systems, USA) as per manufacturer’s instructions. Briefly, plates were pre-coated with capture antibody and then blocked with a universal diluent. Antibody standards were serially diluted in the universal diluent, constructing a 7-point curve with a universal buffer as blank. Cell supernatant (undiluted) was then added in duplicates into the appropriate wells and agitated on a Thermomixer (Eppendorf, Germany) at 800rpm for ≥2 hr at room temperature. Wells were thoroughly washed with 0.05% Tween 20 in 1X PBS (PBST) before the detection antibody was added and agitated for 1 hr at 800rpm at room temperature. After washing, a developing solution with reporter enzyme and substrate was added and agitated for a further 1 hr at room temperature. Absorbance was then recorded at 450nm using a plate reader (CLARIOstar Monochrome Microplate Reader; BMG Labtech, Australia).