*4.3. Cell Death Assays*

A549 lung epithelial cells were seeded at 1200 cells/well in 96-well plates (Celltreat, Pepperell, MA, USA) and allowed to culture for 24 h in Ham's F-12K medium (Thermo Fisher) with 10% FBS (VWR, Radnor, PA, USA) at 37 ◦C and 5% CO2. Following this, cells were washed and the following was added to wells in Ham's F-12K medium: (1) ricin, (2) 100 ng/mL cell death ligand/cytokine (TRAIL, TNF-<sup>α</sup>, or FasL), or (3) ricin combined with 100 ng/mL cell death ligand/cytokine. Cells in negative control wells were treated with media alone. In some experiments (Figure 1B), CHX was used in place of ricin. In experiments where inhibitors were used, they were added to cells 1 h before the addition of ricin and cell death ligands/cytokines. Appropriate vehicle controls were used for each inhibitor. After the addition of ricin and cell death ligands/cytokines, cells were incubated for 24 h at 37 ◦C and 5% CO2. Cell death was then measured using the WST-1 assay (Takara, Kusatsu, Shiga Prefecture, Japan) according to the manufacturer's instructions. Absorbance was measured using an Eppendorf 2200 plate reader at a wavelength of 450 nm and a reference wavelength of 600 nm. Using WST-1 absorbance (abs), percent viability was calculated as follows: (abs cell death stimulus + ricin)/(abs neg) × 100.
