Teto h2bgfp

To specifically label myonuclei via genetic means, we generated female HSA +/--GFP +/-mice by crossing homozygous human skeletal actin reverse tetracycline transactivator (HSA-rtTA) mice developed by our laboratory (Iwata et al., 2018) with homozygous tetracycline response element histone 2B green fluorescent protein mice (TetO-H2B-GFP) obtained from the Jackson Laboratory (005104). GFP labeling is >90%, is highly specific to myonuclei, and does not result in labeling of satellite cell-derived myonuclei during the experimental period (Iwata et al., 2018) , thus making the results specific to resident myonuclei. Mice were treated with doxycycline in drinking water (0.5 mg/ml with 2% sucrose) for one week. Following doxycycline treatment and a six day washout, mice underwent bilateral sham surgery (biological duplicate) or synergist ablation mechanical overload (OV) of the plantaris (biological triplicate) as described previously (von Walden et al., 2020a) , then were euthanized 72 hours later. The mice in these experiments were ~3 months of age at the time of surgery, and immunohistochemistry and single fiber imaging for representative images is described in (von Walden et al., 2020a) . For the metabolic RNA labeling experiments, age-matched C57BL/6J mice were subjected to sham and OV by the same surgeon as the HSA-rtTA mice (biological duplicate for sham and triplicate for OV).