Hank s buffered salt solution hbss

ARPE-19 Tet-ON cells (described previously)50 (link), 67 (link) expressing a doxycycline-inducible version of DHFR.YFP.HA under the CMV/TO promoter were seeded at a density of either 200,000 cells per well of a 24-well plate (for western blotting experiments) or 50,000 cells per well of a 96-well plate (for viability experiments) and allowed to reach confluency over the course of 2 days. Cells were then induced with doxycycline (100 ng/mL, a concentration that does not affect mitochondria biogenesis)⁶⁸ (link) and the indicated concentration of TMP or 14a for 24 h. Cells used for western blotting were then washed with Hank’s Buffered Salt Solution (HBSS, Sigma Aldrich), incubated with fresh media, and harvested at the indicated time point as described earlier. Samples were frozen at −20°C until use. Cells used for viability experiments were treated with doxycycline and TMP or 14a for 24 h, followed by a resazurin mitochondrial reduction potential assay (described previously,⁶⁹ (link) 30 min, 37°C) and CellTiter-Glo 2.0 assay (10–15 min at RT; Promega, Madison, WI, USA).

Protocol #16-025 was reviewed and approved by the Boston University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (N = 10) with body weight 343.8 ± 60.2 g were sedated via intraperitoneal injection of xylazine (10 mg/kg) and ketamine (90 mg/kg). After ensuring appropriate depth of anesthesia and analgesia, animals were euthanized via abdominal aortic exsanguination. The lungs were excised and insufflated via tracheostomy with 10–12 mL of 1.5% low melt agarose (HyAgarose, ACTGene Inc., Piscataway, NJ, United States) in Hanks’ buffered salt solution (HBSS, Sigma) at 37°C, according to previous techniques (Watson et al., 2016 (link)). Excised lungs were then placed on ice for 15 min to allow for solidification of the agarose.