Mouse C2C12 myoblast cells were cultured in growth medium [high-glucose Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin; Gibco)] at 37°C in a 5% CO2 atmosphere. After reaching 80–90% confluence, C2C12 cells were then differentiated into myotubes by replacing growth medium with differentiation medium (high-glucose DMEM supplemented with 2% horse serum and 1% antibiotics) for 4 days.
Palmitate (Sigma-Aldrich; cat. no. P0500) was dissolved in ethanol and diluted to 500 µmol/l in DMEM containing 2% AlbumiNZ bovine serum albumin (MP Biomedicals, Solon, OH, USA; cat. no. 199896), 2% FBS (Atlanta Biologicals, Flowery Branch, GA, USA), 2 mmol/l l-carnitine (Sigma-Aldrich; cat. no. C0283), and 1% antibiotics . Control C2C12 myotubes were incubated in the same medium except that palmitate was substituted with an equal volume of ethanol. In some treatment conditions, 10 µmol/l compound C (prepared in dimethylsulfoxide [DMSO]; Sigma-Aldrich) was coincubated with palmitate for 12 h; DMSO was also used as a vehicle control for the treatments. To inhibit ER stress, C2C12 myotubes were pretreated for 1 h with 1 mM taurourdodeoxycholic acid (TUDCA) (Millipore, Billerica, MA, USA; cat. no. 580549) before adding palmitate for another 12 h. To activate AMPK signaling, the AMPK agonist 5-amino-1-β-d-ribofuranosylimidazole-4-carboxamide (AICAR) (Sigma-Aldrich; cat. no. A9978) or ex229 (Selleckchem; cat. no.S8654) was added to the myotubes at a concentration as indicated in figure legend for different times.
Adenoviral constructs containing short hairpin RNA (shRNA) against AMPKα1 (PRKAA1, NM_001013367) or scrambled shRNA (negative control) were constructed by Genechem (Shanghai, China). C2C12 myotubes were transfected with the adenoviral constructs (multiplicity of infection of 100) for 12 h, and then treated with palmitate or left untreated as described above.
RNA extraction and real-time (RT-) PCR
Total RNA was extracted from C2C12 myotubes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA; cat. no. 15596-026). RNA concentration and quality were verified using a Bio Photometer (Eppendorf, Hamburg, Germany). The PrimeScript RT reagent kit (Takara Bio, Otsu, Japan; cat. no. RR037A) was used to reverse transcribe total RNA (2 µg) into cDNA with random hexamer primers. RT-PCR was performed on a StepOnePlus RT-PCR system (Invitrogen) with fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA; cat. no. 4385612). Each RT-PCR mixture (final reaction volume = 50 µl) contained 21 µl sterile water, 25 µl SYBR Green, 2 µl cDNA (500 ng/µl), and 1 µl each of forward and primers (10 pmol/µl). The reaction conditions were as follows: denaturation at 95°C for 10 s, annealing at the melting temperature of the specific primer set for 15 s, elongation at 72°C for 20 s, and a melting curve step. Target gene expression levels were normalized to that of the 18S rRNA gene. The following forward and reverse primers were used: binding immunoglobulin protein (BIP), 5′-AAACCAAGACATTTGCCCCAG-3′ and 5′-AGACACATCGAAGGTGCCG-3′; CHOP, 5′-CCTAGCTTGGCTGACAGAGG-3′ and 5′-CTGCTCCTTCTCCTTCATGC-3′; ATF4, 5′-GGAATGGCCGGCTATGG-3′ and 5′-TCCCGGAAAAGGCATCCT-3′; growth arrest and DNA damage-inducible protein (GADD)34, 5′-CGGAAGGTACACTTCGCTGA-3′ and 5′-CGGACTGTGGAAGAGATGGG-3′; XBP1s, 5′-GAGTCCGCAGCAGGTG-3′ and 5′-GTGTCAGAGTCCATGGGA-3′; unspliced XBP1 (XBP1u), 5′-AAGAACACGCTTGGGAATGG-3′ and 5′-ACTCCCCTTGGCCTCCAC-3′; and 18S rRNA, 5′-CCAGAGCGAAAGCATTTGCCAAGA-3′ and 5′-TCGGCATCGTTTATGGTCGGAACT-3′.
C2C12 myotubes were lysed in radioimmunoprecipitation assay buffer (Merck, Darmstadt, Germany) with complete EDTA-free protease and phosphatase inhibitors (Roche, Basel, Switzerland; cat. no. 04906845001). The supernatant was collected by centrifugation at 12,000×g for 10 min at 4℃ and the protein concentration was determined using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of extracted protein (30 µg per lane) were denatured with gel loading buffer after centrifugation to remove insoluble material and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane that was blocked in 5% nonfat milk diluted in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h and then incubated overnight at 4°C with primary antibodies against CHOP (cat. no. 5554), ATF4 (cat. no. 11815), AMPKα (cat. no. 2532), p-AMPKα (cat. no. 2531) (all from Cell Signaling Technology, Danvers, MA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; cat. no. sc130656). The following day, the membrane was washed 3 times with TBST and incubated for 2 h at room temperature with secondary antibodies in 5% nonfat milk, followed by incubation with enhanced chemiluminescence reagent (Thermo Fisher Scientific; cat. no. 34580) in a dark room. Protein bands were quantified using Image-Pro Plus v6.0 software (Media Cybernetics, Rockville, MD, USA); the densitometry of the protein signal was normalized to that of β-actin.
Data are presented as mean ± SD. One-way analysis of variance followed by the Bonferroni posthoc test was used to compare the means of multiple groups using Prism v8.0 software (GraphPad, La Jolla, CA, USA). P ≤ 0.05 was considered statistically significant.