Fk506

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).

Cells were cultivated in EMM + E medium overnight and harvested by centrifugation (2,500 rpm, 3 min). Cells were washed by EMM-N medium three times and resuspended in the same medium. Cells (0.3 OD₅₉₅) were cultivated for 24 h at 27°C. Then, cells were inoculated in EMM + E or EMM + S medium with or without nutrients or chemicals. The initial concentration of cells was 0.05 OD₅₉₅ for assays in test tubes or 0.0033 OD₅₉₅ for assays in 96 well plates. Cell growth in 96 well plates was assessed by measuring the turbidity at 595 nm using MULTISKAN FC (Thermo). SLF, FK506, and rapamycin were from Cayman, FUJIFILM, and AG Scientific, respectively. When cells were spotted on solid media, 5- or 10- fold serial dilution series were prepared in EMM-N medium, 5 μL of which were spotted. The highest concentration was 5 × 10ˆ5 cells/mL or 0.015 OD₅₉₅.