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minimum essential medium and Dulbeccos Modified Eagles medium were purchased from Gibco, Foetal bovine serum was purchased from Biosera. Fatty acid-free BSA fraction V was purchased from Roche Applied Science. All other reagent-grade chemicals for buffers, palmitate, phenformin, metformin, salicylate, berberine, quercetin, BSA, and milk were purchased from Sigma-Aldrich unless otherwise stated. Antibodies used and where they were purchased are indicated in Table 1.
Cell culture (carried out by Dr Clare Stretton)
L6 muscle cells were maintained in α-MEM containing 2% (v/v) FBS at 37°C in 5% carbon dioxide. For experiments cells were seeded into 6-well plates and allowed to differentiate into myotubes over a 7-8 day time period before being treated as described. AMPK α1 knockdown cells were generated as described previously (Turban et al., 2012).
HEK Ad293 cells were from the laboratory of C. Sutherland and were maintained in DMEM containing 10% (v/v) FBS at 37°C in 5% carbon dioxide.
Lysate Preparation and Immunoblotting.
L6 muscle cells were allowed to differentiate in 6-well plates then treated with palmitate, berberine hydrochloride and sodium salicylate at times and concentrations stated in the figure legends. After incubation, the muscle cells were rinsed twice in ice-cold PBS (Phosphate-Buffered Saline) and 50μl of lysis buffer was added, all on ice. Cells were scraped and transferred to 1.5ml tubes, lysed by sonication and then centrifuged at 6000 rpm, 4°c for 10 minutes. Supernatant was removed and stored at -80°c until needed.
Cell lysate protein concentration was determined using a Bradford protein assay (Bradford, 1976). 30μg of cell lysate was separated by SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis) on a 10% polyacrylamide gel and transferred onto PVDF (polyvinylidene fluoride) membranes which were then blocked using either 1% fish-skin gelatin (FSG) or 5% non-fat milk, both made up in TBS (Tris-buffered saline) containing 1% (v/v) Tween 20. Membranes were probed over night with primary antibodies against proteins of interest. The membranes were washed for 10 min, 3 times with TBS containing Tween 20, before appropriate secondary anti-Mouse IgG or anti-Rabbit IgG antibody was applied. Protein signals were visualised using the enhanced chemiluminescence technique by exposure to autoradiographic film (Konica Medical Film). Protein bands on film were quantified using ImageJ software (http://rsbweb.nih.gov/ij/).
Adenoviral Infection (carried out by Dr Clare Stretton)
Adenoviruses containing either myc-tagged wild type human IKKβ or a human IKKβ construct in which Ser 670 has been mutated to an alanine have previously been generated in the laboratory. High-titre stocks were generated by infecting flasks of Ad293 for 2h with 200µl of virus stock. Cells were incubated until 50-70% demonstrated cytopathic effect. Cells were scraped into the medium and freeze-thawed four times, then centrifuged at 1000rpm for 5 min to pellet the debris. The virus-containing supernatant was aliquoted and stored at -80°C until required.
Virus was quantified by plaque assay. Briefly, serial dilutions of viral stocks were made up and added, in triplicate, to HEK Ad293 cells in 6-well plates. Cells were incubated for 2h at 37°C then overlaid with DMEM containing 0.6% agarose. Cells were incubated for 7 days before being fixed in 4% (w/v) formaldehyde then stained with napthol blue to visualise plaques.
L6 cells were seeded in 6-well plates and transduced with either IKKβ-expressing adenovirus or an empty adenovirus that does not express any protein at 4 days post seeding. Cells were washed in serum-free medium and transduced with 5 x 106 plaque-forming units of virus stock in a final volume of 500µl serum-free medium. After 2h 1ml complete medium was added to the cells and the cells incubated overnight. Medium was changed on the cells the following day and cells were then incubated for a further two days before being treated as described in figure legends.
L6 cells were seeded on coverslips in 6-well plates and transduced with increasing doses of adenovirus as described above. They were then fixed in 4% paraformaldehyde dissolved in PBS for 10 minutes at room temperature. Coverslips were transferred onto parafilm in petri dishes, where they were permeabilised in 70μl 1% Triton X for 10 min. Coverslips were washed 3 times in 100 μl wash buffer (0.2% BSA/PBS) before a 10% solution of blocking buffer was added to each coverslip (10 μl goat serum made up in 100 μl wash buffer), and left for one hour. Coverslips were again washed 3 times and a 1:500 dilution of primary myc antibody, made up in wash buffer was added and left overnight at 4°c. Coverslips washed for 10 min, 3 times and a 1:500 dilution of secondary antibody, anti-mouse conjugated to Alexafluor 488 (Life Technologies) made up in wash buffer was added and left for 1 h in the dark at room temperature. After a further 3 x 10 min washes, coverslips were mounted onto labelled slides using Vectashield mounting fluid (VectaLabs). Cells were visualised using a Leica DMIRB fluorescent microscope.
RNA Extraction, cDNA Synthesis, PCR and qPCR.
Total RNA was extracted from L6 muscle cells by lysing the cells in 300 μl of TriReagent. 60 μl of chloroform was added to each tube and vigorously shaken for 15 s, before the samples were centrifuged for 15 min, 12,000xg at 4°c. Supernatant was removed and transferred to a new tube containing 150 μl of isopropanol, and left at room temperature for 10 minutes. Samples then centrifuged for 10 minutes at 12,000xg, 4°c and supernatant was removed. 300 μl of RNAase free ethanol was added to the remaining pellet, and spun at 7,500 G for 5 min. Ethanol was removed and eppendorfs were left to air dry for 15 min before 25 μl of RNAase free water was added.
A UV spectrometer was used to quantify the amount RNA present in each of the samples. First strand cDNA was synthesised using M-MLV reverse transcriptase (Promega) as described by the manufacturer. Samples were incubated for 1h at 42°C then 70°C for 10 min to denature the enzyme.
Quantitative real-time PCR was performed using a StepOnePlus Real-Time PCR system (Applied Biosystems), SYBRgreen Jumpstart Mastermix kit (Sigma-Aldrich) and primers targeting IL-6 and GAPDH. The sequences for IL-6 and GAPDH primers are as follows: IL-6 forward, 5′-GACTGATGTTGTTGACAGCCA-3′; IL-6 reverse 5′-ATGCTTAGGCATAACGCACTAGGTT-3′; GAPDH forward, 5′-TGGAAAGCTGTGGCGTGAT-3′ and GAPDH reverse 5′-GCTTCACCACCTTCTTGAT-3. A 1:15 dilution of cDNA for each sample was made up in fresh deionised water. A master mix containing 2 μl of primer and 5 μl of SYBR green mix for each sample was added to a well of a qPCR plate along with 3 μl of diluted cDNA. PCR amplification cycles included initial denaturation at 95°c for 2 min, followed by 40 cycles of denaturation at 95°c for 15 s, annealing at 55°c for 15 s, and extension at 68°c for 30 s. Fold changes in IL-6 mRNA were calculated using the Pfaffi formula (Pfaffl, 2001).
Statistical analysis was performed using a one-way ANOVA/student's t-test. Data analysis was performed using SigmaPlot 12 software and considered statistically significant at values of P < 0.05.