Chir 99021

scAAV8. U7snRNAs particles were produced by Astellas Pharma Inc. (former Audentes). We selected three cell lines containing the largest numbers of repeats: 900, 1,150, and 1,450 CTGs. 7.5E+5 FM cells were plated on 10 cm Petri dishes and 5.5E + 4 FM cells were seeded on glass chamber slides. Once the cells reached 80% confluence, they were transduced with AAVs diluted in Skeletal Muscle Cell Growth medium (Promocell, C23060), supplemented with 5 uM CHIR 99021 (Axon Medchem, 1,386), 10 uM DAPT (Tocris, 2634), 8 ug/mL doxycycline (Fisher Scientific, BP2653-5). A total of 9E + 12vg or 6E + 11vg AAV particles were added to the Petri dishes and glass chamber slides, respectively. The same dose was used for all different AAVs and cells. However, due to limited AAV8. U7snRNA.20CTG volume, 1150CTG FM cells on the Petri dish received approximately half of the AAV amount. Two days after transduction, the growth medium was replaced by Skeletal Muscle Cell differentiation medium (Promocell, C23061), supplemented with CHIR 99021, DAPT, and doxycycline. Medium was refreshed every other day for 7 days when the cells on the glass slides were fixed on 4% PFA and cells on Petri dishes were pelleted and frozen at −80°C. Experimental design is shown in Figure 2B.

FibroMyoD (FM) cells were grown in growth media until cells reached 80% confluency. At that point, growth media was changed to Skeletal Muscle Cell Growth medium (Promocell, C23060), supplemented with 5 uM CHIR 99021 (Axon Medchem, 1,386), 10 uM DAPT (Tocris, 2634), 8 ug/mL doxycycline (Fisher Scientific, BP2653-5), which induces the expression of MYOD and consequent conversion of fibroblasts into myoblasts. After three to 4 days, the cells acquired an elongated and fusiform morphology and became oriented parallel to each other. At this point, cells were washed with PBS, and the myoblast growth medium was replaced by Skeletal Muscle Cell differentiation medium (Promocell, C23061), supplemented with CHIR 99021, DAPT, and doxycycline, to induce the fusion and differentiation of myoblasts into multinucleated myotubes. The medium was changed every other day for 7 days when cells were fixed with 4% PFA for immunofluorescence staining.