IncuCyte system assays

Cells (1 × 10⁵) were seeded in 24-well plates and after 12 hr were transfected with 20 ng/ml of pIC (high molecular weight (1.5–8 kb, InvivoGen) with Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. The cells were incubated with 250 nM Sytox-Green dye (Thermo Fisher), a nucleic acid stain that is an indicator of dead cells and which is impermeant to live cells, and 250 nM of cell permeable dye Syto 60-Red (ThermoFisher), which allows quantification of the total number of cells present in each field, using an IncuCyte Live-Cell Imaging System and software (Essen Instruments 2015A) for 36 hr. Cell death was measured by counting the green objects per mm² (dead cells, green) and then normalizing to the total number of cells per mm² (red objects) at each time point using IncuCyte software. The WT and RNASEL KO HME (2 × 10⁵) cells were grown in 12-well plates and transfected with different concentration of pIC (250 ng/ml, 100 ng/ml, 50 ng/ml and 20 ng/ml). Cell death was measured with the IncuCyte system with Sytox-Green dye (250 nM) for 20 hr.