Synergy h1 gen5

After 72 h of doxycycline induction, 2 × 10⁵ OCI-AML 3 cells/well were seeded onto 12-well plates (Eppendorf, MI, Italy). Viable and dead cell counts were performed with Trypan Blue reagent (Sigma-Aldrich, St. Louis, MI, USA) at the indicated time points, considering 72 h post-induction with doxycycline as the starting point. For cell proliferation curves, only viable cells were considered as part of the total number counted for each well considered. The same assays were performed under treatments with different concentrations of etoposide for the time points indicated. For dose–response cell viability assays, 2 × 10⁴ cells per well were seeded onto 96-well plates (Eppendorf, MI, Italy) once incubated with etoposide treatments at indicated concentrations or vehicle solutions at 72 h post-induction with doxycycline, considered as a starting point. At the indicated time points, cells were incubated for 2 h with CellTiter-Blue Cell Viability assay (Promega, Madison, WI, USA), according to manufacturer’s instructions, and then the plates were acquired with the microplate reader Synergy H1 Gen5 (Biotek, Agilent, Santa Clara, CA, USA).

BY4741 or ADH1-knockout cells transformed with plasmids were cultured in hypoxic conditions as described above. Each strain was cultured in three wells of a 24-well plate in all experiment. After 6 h, yeast cells were collected, fixed, and resuspended in 1 mL of 1× PBS. Cell suspensions from single wells of 24-well plates were transferred to 96-well plates (CellCarrier^™-96 ultra, PerkinElmer Japan Co., Ltd., Yokohama, Japan) in 100 μL aliquots in triplicate. 1 ×PBS was used as a negative control. The fluorescence intensity of FusionRed was measured using a fluorometric plate reader (Synergy H1 (GEN5); BioTek, Winooski, VT, USA) equipped with a Red FP filter cube (Excitation filter 530/25, Barrier filter 590/35, Dichroic mirror 570 nm; BioTek). OD₆₀₀ values were measured using a Molecular Devices Spectra MAX 190 microplate reader (Moleular Devices, CA, USA). The FusionRed intensity of each strain was calculated as fluorescence per OD₆₀₀ (FusionRed fluorescence/OD₆₀₀) using the following formula:
$$\text{Fluorescence}\left(\text{sample}\right)-\text{fluorescence}\left(\text{blank}\right)/({\text{OD}}_{600}\left(\text{sample}\right)-{\text{OD}}_{600}\left(\text{blank}\right)$$