LNA treatment, caspase activation and cell viability assays

All cell-lines used for in cellulo experiments, Vero E6, and Huh-7, and ACE2-A549 were maintained at 37°C in complete Dulbecco’s modified Eagle medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Invitrogen), penicillin and streptomycin (Gibco), and HEPES buffer (Gibco). The ACE2-A549 cells were specially engineered to over-express the ACE2 receptor in the human alveolar basal epithelial cells, A549.

For caspase activation and cell viability assays, one day prior to transfection, 10,000 cells per well were plated in growth media in black-walled, clear bottom 96-well plates (EK-25090, E&K Scientific) to be 70% confluent at time of transfection. 24 h later, LNA-modified ASOs were diluted in Opti-MEM I and mixed with Lipofectamine 3000^® reagent (Life Technologies) according to manufacturer′s protocol to the indicated final concentrations. The complexes were incubated at room-temperature for 30 mins, after which 10 μL of each of the LNA-Lipofectamine complex was added dropwise to cells containing 90 μL of growth media. Cells were then incubated as described. A caspase-activating LNA, Pos_LNA, and known apoptotic activator, camptothecin, were used as positive controls.

To test caspase activation, 24 h post-transfection with the LNA ASOs, the Caspase-Glo 3/7 reagent (G8092/G8093 kit; Promega, Madison WI, USA) was prepared at room-temperature according to the manufacturer′s specifications. Once the cell plates were equilibrated at room temperature, a 1:1 ratio of Caspase-Glo 3/7 reagent volume to sample volume was added, its contents were mixed gently at 350–500 rpm for 30 s using a plate shaker, and left to incubate at room temperature for 45 mins in the dark. The plates were measured for caspase activation by luminescence using a Tecan Infinite® multimode plate reader (M1000, Tecan). Data were analyzed and graphed in Prism 8 by GraphPad.

Cell viability after LNA ASO treatment was determined by PrestoBlue cell viability reagent (Invitrogen) following the manufacturer′s instructions. LNA ASO transfections were generated using the same transfection protocol described above in 96-well plates. After 24 h post-transfection, the media was replaced with complete DMEM and cells were incubated for an additional 3 days. 10 μl of Prestoblue reagent (catalog no. 11644807; Roche Applied Science) was then added to each well and the plate was incubated for 1 h. The plate was then read for fluorescence (540 nm excitation/590 nm emissions) using a Tecan Infinite® multimode plate reader (M1000, Tecan). The cell viability after 4-days of LNA ASO treatment was calculated as a percentage of total cellular viability, normalized to the average viability count of non-treated wells in the absence of LNA ASOs after background correction. Data were analyzed and graphed in Prism 8 by GraphPad.