Mini spin column

Mitochondrial DNA content was determined as described previously (Zunica et al., 2021 (link)). Briefly, 15 thoraces per biological replicate were isolated, suspended in PBS (pH 7.2; 50 mM potassium phosphate, 150 mM NaCl), and homogenized. Endogenous nucleases were removed via incubation in proteinase K, followed by ethanolic extraction and elution in a mini spin column (Qiagen, Cat # 69504). Total DNA content was quantified (NanoDrop), normalized to 10 ng of DNA, and assayed by qPCR for the mitochondrial DNA to nuclear DNA ratio with primers targeting Mt:Col and Cdk4 (Table S1).

In accordance with the manufacturers’ protocols, Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and RNeasy mini kits (Qiagen, Valencia, CA, USA) were used for the extraction of total RNA. Tri-reagent was used to homogenize muscle and the subsequent extraction was performed in phenol/chloroform. Seventy percent ethanol was added to the aqueous phase and the sample was then applied to a Qiagen minispin column, and the protocol from the manufacturer was followed [20 (link)]. RNA was eluted from the column using RNase-free water and then quantified (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was assessed using 1% agarose gel, and total RNA was reverse-transcribed (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. An aliquot of the reverse-transcribed reaction mix was used for qPCR and TaqMan gene expression assays: CAT (cationic amino acid transporter)-1 (NM_013111.2), SNAT (sodium-coupled neutral amino acid transporter)-2 (NM_181090.2), LAT (large neutral amino acid transporter)-2 (NM_019283.1), PAT (proton-coupled amino acid transport)-1 (NM_130415.1), PAT-2 (NM_139339.1), and PAT4 (NM_001108127.1) (Applied Biosystems, Foster City, CA, USA). The endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH, NM_017008.3) was used in the expression of target genes by the 2^−ΔΔCt method.

Genomic DNA of each bacterial sample was extracted using a QIAamp®DNA Minikit (#51304, Qiagen) according to the user manual. In brief, bacterial cells were collected from a 1 mL sample by centrifugation at 7500 rpm for 5 min. The pellets were suspended in 180 µL of an enzyme solution (20 mg/mL lysozyme in 20 mM Tris–HCl, 2 mM EDTA, and 1.2% Triton-X 100) and incubated at 37 °C for 1 h. The pellets were treated with 20 µL of proteinase K at 56 °C for 2 h before being treated with 4 µL of RNase A (100 mg/mL) at room temperature for 2 min. The genomic DNA was further purified by a Qiagen mini spin column eluting with 50 µL of sterile water. Concentration and purity of the obtained genomic DNA were determined by UV-spectrophotometry (NanoDrop 8000 spectrophotometer, USA) at 260 and 280 nm.