C2c12 myoblasts

HEK293 (ATCC, CRL-1573), HEK293T (ATCC, CRL-3216), MDA-MB-231 (ACC 732) and Hep3B (HB-8064) cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and American Type Culture Collection. C2C12 myoblasts were purchased from Sigma Aldrich (ECACC 91031101). Cells were cultivated in DMEM, 10% fetal calf serum.

C2C12 myoblasts (catalog no. 13K011, Sigma) were cultured on 10 cm gelatin-coated plates. The surface of the plate was coated with 0.2% gelatin in double-distilled H₂O that was then discarded after a few minutes. The plate was allowed to dry for 1–2 h in the laminar tissue culture. Cells (1 × 10⁶/10 cm dish) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; catalog no. 12–604 F, Lonza) with 20% fetal bovine serum (FBS; catalog no. E5050–02, lot no. 70932, EURx), 0.1% fungizone, and 1% penicillin/streptomycin. Different lots of FBS from the same suppliers may vary and can affect AChR clustering. Therefore, testing different lots of FBS and selecting the one that efficiently facilitates the clustering of postsynaptic machinery are important. At all stages of culturing, the cells were evenly spread on the dish to prevent dedifferentiation. Cells were passaged every 3 days and before they reached 30% confluence (see Fig. 2 for recommended densities). After trypsinization, the cells were collected by centrifugation at 419 g for 3 min, resuspended in fresh culture medium, and split according to a 1:6 ratio onto fresh gelatin-coated dishes that contained warm culture medium. Cells that are passaged this way take ~2 days to reach 30% confluence.

C2C12 myoblasts (Sigma) were grown and induced to differentiate into myotubes as described [58 (link)]. Stable cell lines ectopically expressing Flag-tagged Cdc73, Rtf1, and Ski8 were generated by cloning cDNAs from Open Biosystems into pBabePuro as described [59 (link)]. For transient transfection of siRNA, cells were seeded at 10⁴ cells cm⁻² for 48 h transfection. 5 μl RNAiMax in 2 ml medium and 50 nM siRNA were applied to cells by using fast-forward method. siRNA target sequences are listed below.
siPaf1: GCUAUGAGGAGAACUAUUU
siCdc73: GAUCCUACAUUGCGUACAA
siSki8: GGUGUUGAAUGUUGCGUUC

iPax7 cells were generated as described (Darabi et al., 2011). iPax7 cells were grown on 0.1% gelatin-coated culture plates in GlutaMAX supplemented IMDM (Gibco) with 15% stem cell qualified fetal bovine serum (Gemini), 200 μg/ml bovine holo-transferrin (Sigma), 10 μg/ml ascorbic acid, 4.5 mM 1-thioglycerol, 100 IU penicillin, and 100 μg/ml streptomycin. Media was changed every two days with the addition of 5 ng/ml recombinant mouse FGF basic protein (R&D systems), and 0.75 μg/ml doxycycline (Dox) for the expression of Pax7. As indicated, iPax7 cells grown in -Dox condition were washed with PBS and switched to media lacking Dox. C2C12 myoblasts (Sigma) were grown in DMEM (cat #15-013-CV Corning) supplemented with 100 IU penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine and 10% fetal bovine serum, and differentiated to myotubes by incubating cells with DMEM supplemented with 2% horse serum. Stable cell lines ectopically expressing carboxy-terminal 3xFlag-tagged Pax7 or 3xFlag-only control were generated as described [35 (link)]. Pax7 cDNA was sub-cloned from p2lox Pax7 into pBABE-puro (Addgene) and constructs were transfected into Phoenix-Eco cells. Harvested viral supernatant was used to transduce C2C12 myoblasts together with polybrene and selected with puromycin (4 μg/ml).

C2C12 myoblasts (Public Health England sourced from ATCC) at passages 3–12 were maintained in basal Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, United Kingdom) supplemented with 20% v/v fetal calf serum [First Link (UK) Ltd., United Kingdom] and 1% v/v penicillin–streptomycin (Gibco Life Technologies, United Kingdom). All cell cultures were kept in a humidified incubator at 37°C and 5% CO₂ for the duration of the experiment.

C2C12 myoblasts were purchased from Bioresources Center (Tsukuba, Japan) and maintained and proliferated at 37 °C with 5% CO₂ in Dulbecco's modified eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, Sigma), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Nacalai Tesque, Kyoto, Japan). At a confluence of 80%, C2C12 myoblasts were transferred to next generation by 0.25% trypsin (Sigma)/0.02%EDTA⋅2Na.