Cell countess

About 2 h prior to cell seeding, the PBS contained in the substrates (glass bottom dish, MEA or well plate) was replaced with 1 mL of NBD. The substrates were placed in an incubator (37 °C, 5% CO₂, Steri-Cycle 371 CO₂ Incubator, ThermoFisher Scientific) until seeding.
An iNeuron aliquot was taken out of the liquid nitrogen and put at 37 °C to thaw rapidly. The 1 mL thawed cell solution was transferred dropwise into 4 mL of warm NBD and centrifuged for 5 min at 1000 rpm. The supernatant was aspirated and cells were resuspended at a concentration of 1 × 10⁶ cells per mL. The cell solution was passed through a 40 μm strainer (CSS013040, BioFilJet) and counted using a cell counter (Cell Countess, Invitrogen).
A volume containing the target cell number was pipetted onto the substrate (30 to 65k cells per cm²). After 10 min, the solution was mixed by pipetting to increase the number of iNeurons in the PDMS nodes. A complete medium exchange was done 1 h after seeding to remove dead cells. For the laminin-supplemented experiments, laminin was added to the medium at this stage, to a final concentration of 1 to 10 μg mL⁻¹. A half medium change was performed two to three times a week, with optional addition of laminin to the medium during the first week of medium change. In all experiments, the day of iNeuron thawing and seeding was considered as DIV 0.

MV4-11 AML cells were seeded onto 96-well plates and kept at a concentration of 0.5 million cells/mL. To determine the cell concentration, the cells were stained with Trypan Blue (0.4% Life Technologies, Carlsbad, CA, USA) and counted using the Cell Countess Invitrogen system. Cells were treated with Magnolia grandiflora extracts at concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, and 40 g/mL, in duplicate. After 48 h of treatment, cell viability was determined by flow cytometry. The cells were stained with YO-PRO-1 (Invitrogen-Thermo Fisher, Waltham, MA, USA) and 7-aminoactinomycin (7-AAD, Invitrogen-Thermo Fisher, Waltham, MA, USA) to assess viability. At least 10,000 events were recorded per condition on an LSR-Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was conducted using the FlowJo 9.6 software for Mac OS X (TreeStar, Woodburn, OR, USA). Cells that were negative for YO-PRO-1 and 7-AAD were scored as viable. This same process was repeated with compounds 1–5, except that viability was determined after treatment by 36 h. All compounds, except for compound 3, were tested at concentrations of 1.25, 2.5, 5, 10, and 20 µM. Compound 3 was tested at concentrations of 1.25, 2.5, 5, 10, 12.5 20, 25, 50, 100, and 200 µM.

The cells were assessed for viability after control treatment or dosing with 200-proof 50 mM and 100 mM EtOH for 48 and 72 h. All media was removed. The cells were gently scraped with a cell scraper with 1.5 mL of non-sterile Phosphate Buffered Saline (PBS) solution. The solution was collected and gently mixed in 2.0 mL Eppendorf tubes. The cells were then counted and viability was assessed using trypan blue dye in Cell Countess (Invitrogen, Carlsbad, CA, USA).

24 hours after transfection, 2.5x10⁴ (for 293T, Rae1 over-expression) and 3.5x10⁴ (for HeLa, Rae1 over-expression) cells were added to each well of multiple12-well cell culture dishes. For the Rae1 knockdown experiment, 1.0x10⁵ cells (for 293T, Rae1 knockdown) were added to each well of multiple 6-well cell culture dishes.
Cells were incubated at 37°C until harvest. At approximately 24, 48, and 72 hours post-seeding, one dish was retrieved from incubation and the contents of each well were aspirated. Each well was washed twice in 1x PBS. The adherent cells were dissociated with 0.05% Trypsin/EDTA and subsequently counted using a Coulter Counter (Beckman) or using Cell Countess (Invitrogen).