Fig 1: mRNA and protein levels of genes involved in cell-death pathways in primary mouse neurons and mouse brains. (A,B) The hACE2 neurons were infected with SARS-CoV-2 or mock-infected at a MOI of 0.1. At 48 h after infection, cell pellets were collected, and total RNA was extracted. qRT-PCR was conducted to determine the fold-change in (A) ZBP1, MLKL, RIPK3 and RIPK1, and (B) Caspase 8, Caspase 1, Caspase 7 and Caspase 8 mRNA levels. Data for each sample was normalized to the value for GAPDH and expressed as the relative fold increase compared to mock-infected controls. Data represent the mean ± SEM of three independent infection experiments conducted in duplicate. Each data point represents an independent experiment. (C) The hACE2 neurons were infected with SARS-CoV-2 or mock-infected at a MOI of 0.1. At 24, 48 and 72 h, cell pellets were collected, and total protein was extracted. Protein was blotted with ZBP1, pMLKL or ß-actin antibodies. Data are representative of three independent experiments. (D,E) K18-hACE2 mice were inoculated with SARS-CoV-2 or PBS via the intranasal route. Brains were harvested after extensive perfusion at days 1, 3 and 6 after infection and RNA was extracted. qRT-PCR was used to determine the mRNA levels of (D) ZBP1, MLKL, RIPK3 and RIPK1, and (E) Caspase 8, Caspase 1, Caspase 7 and Caspase 8. After normalizing individual sample to GAPDH level, the fold change in infected tissues compared to mock-infected controls was determined. Each data point represents an individual mouse (n = 4). Values are the mean ± SEM. (F) Protein extracted from mock- and SARS-CoV-2-infected brain tissues were blotted with ZBP1, pMLKL or ß-actin antibodies. Data are representative of four mice per time point.