C2c12 cells

C2C12 and HEK293T cells were purchased from ATCC (Manassas, VA) and were maintained at subconfluent densities in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator at 37°C in 5% CO₂. For differentiation, C2C12 cells at >80% confluence were switched to DMEM supplemented with 2% horse serum and 2 μg/ml of bovine insulin (Sigma-Aldrich). FK506 (Cayman Chemical, Ann Arbor, MI) was added to the culture 24 h before initiation of differentiation, and FK506 was maintained in the differentiation medium at 2 μM. Medium containing FK506 was changed every day.
Mouse satellite cells were isolated from leg muscles of 3-to-6-week-old Brg1 conditional mice by the use of Percoll sedimentation followed by differential plating as described previously (20 (link)). Mice were housed in the animal care facility at the University of Massachusetts Medical School and used in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Brg1-depleted primary myoblasts expressing wild-type Brg1 (WT-Brg1) or Brg1 mutated at sites of PKCβ1/Cn activity were generated as described previously (20 (link)). Primary myoblasts were grown and differentiated as described previously (20 (link)) on plates coated overnight in 0.02% collagen (Advanced BioMatrix, San Diego, CA).

Skeletal muscle (C2C12) cells (Sigma Aldrich, Melbourne, Australia) were cultured in 10% fetal calf serum, and 1% Penicillin-Streptomycin-supplemented DMEM containing 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma Aldrich, Melbourne, Australia) and grown in an optimal condition of 5% CO₂ at 37 °C. Once the cells reached 70% confluency, the C2C12 skeletal muscle myotubule phenotype was obtained by mitogen withdrawal (2% horse serum, Thermo Fisher, Melbourne, Australia) for 72 h. Three hours before the treatment, the C2C12 cells were exposed to a serum-free medium. Cells were then treated with various combinations of capsaicin, insulin, AICAR, and SB-452533 as per the following procedures.

C2C12 cells (Korean Type Culture Collection, Seoul, Korea) were cultured in DMEM medium containing 10% fetal bovine serum, 1% antibiotic–antimycotic solution, and 3.7 g/L NaHCO₃ at 37^oC in a 5% CO₂/95% air humidified incubator [16 (link)]. Confluent myoblasts (80%) were differentiated in DMEM medium containing 2% horse serum for 4 days. Probiotics (1 × 10⁴ cells/mL) or creatine (Cr, 5 mM, C0780, Sigma) were treated 10 h after treatment with dexamethasone (10 µM, D4902, Sigma) or lipopolysaccharide (LPS, 100 ng/mL) in differentiated C2C12 cells (1 × 10⁵ cells/mL).

C2C12 cells were obtained from Sigma (#91031101) along with Dulbecco's Modified Eagle Medium (DMEM; #D6429), fetal bovine serum (FBS; #F0926), horse serum (#H1270), and insulin (#I9278). Recombinant IL-15 was from GenScript (#Z03309-50) and GW-6471 (#11697) and GSK-3787 (#15219) were obtained from Cayman Chemicals. Trizol (#15596), SYBR green (#A25742) and a SuperScript VILO reverse transcription kit (#11754050) were purchased from ThermoFisher. The mitochondrial dye, Mito Red, was from Santa Cruz Biotechnology (SC-#301164). Male C57BL6 mice were kept on a 12 : 12 light dark cycle and fed a standard diet and water ad libitum. Gastrocnemius muscle was obtained following euthanasia. All animal procedures were approved by the Institutional Animal Care and Use Committee at Chapman University.