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).