Antibiotic antimycotic mixture

The mitochondrial bioenergetics stress test was conducted to measure mitochondrial oxygen consumption rates (OCRs) using a Seahorse XFe24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA) (Abdullah et al., 2019 (link); Aishwarya et al., 2020 (link); Alam et al., 2018 (link)). Neonatal rat ventricular cardiomyocytes (NRCs) were plated at a density of 8 × 10 ⁴ cells per well into 0.1% w/v gelatin-coated Seahorse XFe24 plates with α-MEM culture media containing 10% FBS with 1% antibiotic-antimycotic mixture (Gibco). After 24 hours of plating, cardiomyocytes were transfected with Sigmar1 siRNA and control siRNA for 16 hours. NRCs were subsequently cultured in DMEM-Glutamax™ culture media containing 2% FBS with 1% antibiotic-antimycotic mixture (Gibco) for 5 days. On day 5, NRCs were incubated with DMEM (containing no glucose and no pyruvate; Gibco) containing 10 mM glucose and 2 mM sodium pyruvate in a CO₂ free incubator at 37 °C for 1 hour before loading the plate in the XFe24 analyzer. The OCRs were recorded over 90 minutes following consecutive injections of oligomycin (1 μM), FCCP (4 μM), and rotenone (0.5 μM) plus antimycin A (0.5 μM) to each well at the given time points. Following completion of the test protocol, total protein concentrations were measured in individual wells to normalize the OCR values as expressed as pmoles/min/μg protein.

Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Invitrogen (Carlsbad, CA). All primary antibodies were purchased from Cell Signaling (Beverly, MA) and Santa Cruz Biotechnology (Dallas, TX). B16F10 mouse melanoma cell line and A375 human melanoma cell line were acquired from the American Type Culture Collection (ATCC). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), and 1.0% antibiotic/antimycotic mixture (Invitrogen) PC3 and PC3-TXR cells were kindly provided by Dr. Evan T. Keller from the University of Michigan. These cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified incubator containing 5% CO₂ at 37 °C and their resistance to taxane was maintained by adding 200 nM paclitaxel to growth media biweekly. 2,2-Bis(hydroxymethyl)propionic acid, methoxy poly(ethylene glycol) (mPEG, 5,000 Da), 8-diazabicycloundec-7-ene (DBU), benzyl bromide, L-lactide, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used as received.

Tissue culture dishes (35-mm) were purchased from Corning Life Sciences (Tewksbury, MA). The Dulbecco's Modified Eagle's Medium, CO₂-independent medium, fetal bovine serum, phosphate buffered saline (PBS), and antibiotic-antimycotic mixture (penicillin, streptomycin, and amphotericin B) were from Invitrogen Life Technology (Invitrogen, Carlsbad, CA). The picric acid and sirius red dye were obtained from Sigma-Aldrich (St. Louis, MO). The PI3K/Akt inhibitor LY294002 and antibodies against total and phosphorylated forms of Akt (Ser473) and Erk1/2 (Thr202/Tyr204) were from Cell Signaling (Beverly, MA). Dihydrorhodamine (DHR) was from Calbiochem/Millipore (San Diego, CA).