Phosphate buffer

AML and MDM cells were fixed with 4% formaldehyde and 1% glutaraldehyde in phosphate buffer (Invitrogen) overnight at 4°C. The samples were processed and imaged using a JEOL 1400 TEM. Expanded protocol is in the detailed supplemental methods section.

PTX and FKA were purchased from Sigma (St. Louis, MO, USA). Sodium aescinate (Aes) (≥98%) was purchased from Meilunbio^® (Dalian, China). Monoclonal rabbit anti-human P-gp (cat. no. 13978) was purchased from Cell Signaling Technology (MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Annexin V‑fluorescein isothiocyanate/propidium iodide Apoptosis Detection Kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The cell cycle detection kit was purchased from Sigma (St. Louis, MO, USA). Dialysis membranes (MWCO 1000) were obtained from the Shanghai Medical Chemical Reagent Co., Ltd. (Shanghai, China). Phosphate buffer and 0.25% trypsin were purchased from Gibco, Invitrogen Corp (Ontario, USA).

The training and validation datasets came from two different male rhesus macaques. The former received an injection of the anterograde/bidirectional tracer Lucifer Yellow in the anterior frontal cortex (frontal pole). The latter received an injection of the anterograde/bidirectional tracer Fluorescein in the ventrolateral prefrontal cortex (vlPFC). Surgery and tissue preparation were performed at the University of Rochester Medical Center. Details of these procedures were described previously (Haber, 1988 (link); Lehman et al., 2011 (link); Safadi et al., 2018 (link)). Briefly, each monkey received an injection of a bidirectional tracer conjugated with dextran amine (40–50 nl, 10% in 0.1 M phosphate buffer, pH 7.4; Invitrogen). Twelve days after the injection, animals were perfused and their brains were postfixed overnight and cryoprotected in increasing gradients of sucrose (10, 20, and 30%). All experiments were performed in accordance with the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals and approved by the University of Rochester Committee on Animal Resources.

Drug stability in plasma was evaluated by incubating 5 μM PY10, PY109, PY108, or ITE in mouse plasma for 0, 5, 15, 30, 45, and 60 min at 37°C with constant shaking at 225 rpm. Reactions were terminated by the addition of twofold (v/v) acetonitrile, followed by centrifugation at 14,000 rpm for 15 min to remove particulates, and the supernatant was analyzed by LC-MS/MS.
Drug metabolic stability was examined using a liver microsomal assay (45 (link)). Briefly, 5 μM PY10, PY109, PY108, or ITE was added to pooled mouse liver microsomes (0.5 mg/ml) in 100 mM phosphate buffer containing MgCl2 (Thermo Fisher Scientific). The assay was activated by the addition of an NAPDH (reduced nicotinamide adenine dinucleotide phosphate)–regenerating system containing NADP⁺, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase (Sigma-Aldrich). Samples were incubated for 0, 5, 15, 30, and 45 min at 37°C, with constant shaking (225 rpm). Reactions were terminated by adding twofold (v/v) acetonitrile, samples were centrifuged at 14,000 rpm for 15 min, and the supernatant was analyzed using LC-MS/MS. t_1/2 values were calculated according to the equation $t_{1/2}=-0.693/k$ where k is the elimination rate constant, obtained by fitting C = initial × exp.^{−k × t}.

Cells were exposed to DBD plasma and after 24h incubation at 37 °C, 50 μM Amplex® red reagent and 0.1 units/ml horseradish peroxidase in phosphate buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were added to each well to a final volume of 100 μl, and the cells were incubated for 30 min in the dark. Fluorescence was assessed at excitation/emission values of 485 nm/580 nm in a microplate reader.