Celltiter glo luminescent reagent

Siomycin A, an antibiotic thiazole compound (FOXM1 inhibitor) (cat no: sc-202339, Santa Cruz., USA) was reconstituted in dimethyl sulfoxide (DMSO) and stored at −20 C. Cells were plated overnight prior to drug treatment and treated at concentrations indicated in each experiment. Cell viability was assessed five days post drug treatment through quantification of ATP levels (CellTiter-Glo luminescent Reagent, Promega, Madison, WI). For siRNA mediated FOXM1 inhibition, 2-pmol siFOXM1 (FlexiTube GeneSolution GS2305 for FOXM1 (contains 4 validated siRNAs for FOXM1) (Qiagen Inc., Germantown, MD) was complexed with RNAi Max lipid transfection reagent (Invitrogen) in DMEM media for 15 minutes at room temperature. Cells suspended in DMEM supplemented with 20% FBS were then added. Plates were maintained at room temperature for 15 minutes before being placed at 37 C/5% CO2. Negative (All star siNegative [siNeg], Qiagen) and positive (All star siCelldeath, Qiagen) control siRNAs were used as controls. 48 hours post transfection cells were processed as indicated.

For the dose response curves, 1800 cells were plated in 36 μl per well of a 384 well plate on day one. Drugs were dissolved in DMSO and a 12 point, two-fold series was prepared. The drugs were then dissolved 1:33 in media and 4 μl was added to each well of the plates on day two. After 24-48 hours of drug treatment (based on the cell line), the viability of cells was measured using a 1:1 dilution of the CellTiter-Glo Luminescent reagent (Promega, cat# G7573) with media, which was read on a Victor 5 plate reader after 10 minutes shaking at room temperature. The intensity of luminescence was normalized to that of the DMSO control. Experiments were performed twice in duplicates each time.
For viability assays when the experiment was performed in 6-well plates, cells were harvested using trypsin (0.5 ml per well) and the media was saved from each well. The trypsinized cells were resuspended with the saved media and 2-3 aliquots (0.05 ml each) sampling different regions of this suspension were transferred into a 96-well plate to serve as technical replicates for the measurement. CellTiter-Glo Luminescent Viability assay was used to measure the viability of these aliquots. The rest of the cultures were used to extract protein to be analyzed using western blots.

For cell death assays^{7 (link),9 (link),11 (link),42 (link)}, cells were trypsinized, collected and stained with trypan blue. The cells were counted with a hemocytometer using the standard protocol, and cells stained blue were considered as dead cells. The cell death was further confirmed by propidium iodide staining followed by FACS analysis. For CellTiter-Glo luminescent assay^{43 (link)}, 10⁴ cells were plated 90 µL per well of a 96-well plate on day 1. The treatments were added to each well of the plates on day 2. The viability of cells was measured using 1:1 dilution of the CellTiter-Glo luminescent reagent (Promega G7572), which was read on a Glomax explorer (Promega) after 10 min of shaking at room temperature. The intensity of luminescence was normalized to that of DMSO control. Percentage of Cell death was counted as 100 minus percentage of cell viability.

Cell proliferation was measured with an impedance-based instrument system (xCELLigence, Roche/ACEA Biosciences, Basel, Switzerland) enabling label-free real time cell analysis. Briefly, 2-4 × 10⁴ cells were seeded into 96-wells with 200 μl culture media containing FBS and allowed to grow up to 150 h. Cellular impedance was measured periodically every 4 h (relative cell index) and HDACi, EEDi, chemotherapeutics or DMSO was added as indicated in the Results section.
For synergy evaluation 0.5-1 × 10⁴ cells were seeded into white, flat-bottom, 96-well plates (Corning). Twenty-four hours later, doxorubicin and/or HDACi (FK228 or MS-275) were added, and viability was assessed after 72 h. Cell viability was measured using CellTiter-Glo Luminescent Reagent according to the manufacturer’s protocol (Promega). Luminescence was measured on a Promega GlowMax-Multi Detection System. Synergistic interactions were evaluated using the package Synergyfinder [30 (link)] (Package version 2.2.4, R version 4.0.3, RStudio version 1.3.1093) according to the Bliss algorithm [31 ].