siRNA-based knockdown of host kinases
(Institut Pasteur, Paris). Host kinases were knocked-down in A549 cells stably expressing ACE2 cells using OnTargetPlus siRNA SMARTpools (Horizon Discovery). Briefly, A549-ACE2 cells seeded at 2x104 cells/well in 96-well plates 18 hours prior to the experiment. The next day, each well was transfected with 6 pmoles of siRNA SMARTpool, using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. Twenty-four (24) hours post transfection, the cell culture supernatant was removed and replaced with virus inoculum (MOI of 0.1 PFU/cell). Following a 1 hour adsorption at 37°C, the virus inoculum was removed and replaced with fresh Opti-MEM media (Thermo Fisher Scientific) containing 2% FBS (v/v). Seventy-two (72) hours post-infection (p.i.), the cell culture supernatant was heat-inactivated at 95°C for 5 minutes, and detection of viral genomes performed by RT-qPCR.