Ap1903

A total of 20,000 HEK293T cells were plated into each well of a poly-l-lysine–coated 96-well flat-bottom plate and allowed to adhere overnight. A constitutive cytokine receptor vector (2.5 ng), a STAT response element vector that drives Firefly luciferase (100 ng; Promega, Madison, WI) and Renilla luciferase control reporter vector (1 ng; Promega, Madison, WI) were mixed in a final volume of 5 μl in Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA) (“DNA mix”). Lipofectamine 2000 (0.3 μl; Invitrogen, Thermo Fisher Scientific, Waltham, MA) in 5 μl of Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mix. The mixture was incubated at room temperature for 20 min, and the total volume of 10 μl was added to each well containing HEK293T cells. Twenty-four hours after transfection, cells were left untreated, treated with AP1903 (1 μg/ml; APExBio, Houston, TX), or with the indicated cytokines diluted in serum-free media. At the indicated time points after treatment, reporter activity was determined using the Dual-Glo Luciferase Assay System (Promega, Madison, WI) as per the manufacturer’s instructions. Firefly luciferase activity was first normalized to Renilla luciferase activity, and then STATresponse element fold induction was calculated by normalizing to untransfected controls.

We used previously described 293T-based landing pad cells to create cell clones that express different levels of ACE2 by modifying the Kozak sequence controlling translation of the gene [37 (link)]. Prior to modification, these HEK 293T LLP-Int-BFP-IRES-iCasp9-Blast clone 3 landing pad cells [37 (link)] were maintained in D10 growth media (Dulbecco’s Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine) supplemented with 2 μg/mL doxycycline and 10 μg/mL blasticidin. These landing pad cells were modified with ACE2 transgenic sequences by transfecting 600,000 cells with 1,200 ng of Kozak-variable AttB_ACE2-miRFP670_IRES_mCherry-H2A-P2A-PuroR recombination plasmid mixed with 5 μL of Fugene 6 reagent per 6-well. The Kozak sequences preceding ACE2 were GCCACCATG, TATCTAATG, TATTTCATG, and AATTTTATG corresponding to “high”, “medium”, “low”, and “very low” cells, respectively [12 (link)]. The cells were transfected in D10 + doxycycline growth media until day 3 after transfection, when AP1903 (ApexBio, B4168) was added to a final concentration of 10 nM to kill off unmodified landing pad cells. Once the cells reached ~ 10% confluence, the cells were switched to D10 + doxycycline growth media containing 1 μg / mL puromycin to achieve a roughly pure population of ACE2 transgenic cells.

HEK 293T cells were used to generate the lenti-landing pad line derived from LLP-Int-BFP-IRES-iCasp9-Blast as previously described [10 (link)]. Landing pad cells expressing Bxb1 integrase with a nuclear localization signal to allow for transport into the nucleus were recombined in either 24-well or 6-well plates. In the 24-well plate, 120,000 cells were transfected with 254 ng of attB recombination plasmid mixed with 0.96 μL of Fugene 6 reagent in D10-dox media. In the 6-well plate, 600,000 cells were transfected with 1,200 ng of attB recombination plasmid mixed with 5 μL of Fugene 6 reagent in D10-dox media.
Upon attB-plasmid transfection, negative selection of nonrecombined landing pad cells was performed with the addition of 10 nM AP1903 (ApexBio, B4168) to activate iCasp9. Positive selection of recombined cells was achieved with the addition of 1 μg/mL puromycin (InvivoGen, ANTPR1). Recombined cells were maintained in D10-dox with 1 μg/mL puromycin to prevent transgene silencing.