Journal: bioRxiv
Article Title: Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2
doi: 10.1101/2021.04.15.440067
Figure Lengend Snippet: (A) HeLa-ACE2 were treated with 20 μM Rap, Eve, Tem, Rid, or an equivalent volume of DMSO for 4 hours. HIV-CoV-2 S pseudovirus incorporating BlaM-Vpr (HIV-BlaM-CoV-2) was added to cells for 2 hours and washed. Cells were incubated with CCF2-AM for an additional 2 hours and fixed. Cleaved CCF2 was measured by flow cytometry. Dot plots visualized as density plots from one representative experiment are shown on the left and the percentage of CCF2+ cells which exhibit CCF2 cleavage is indicated. Summary data representing the average of four experiments is shown on the right. (B) HIV-CoV-1, (C) HIV-MERS-CoV, (D) HIV-IAV HA, or (E) HIV-VSV G were added to HeLa-ACE2 or HeLa-DPP4 cells as in (A) and infection was measured by luciferase activity at 48 hours post-infection. Luciferase units were normalized to 100 in the DMSO condition. Means and standard error were calculated from 3–4 experiments. Statistical analysis was performed with one-way ANOVA and asterisks indicate significant difference from DMSO. *, p < 0.05; **, p < 0.01. Rel.; relative.
Article Snippet: pcDNA3.1 encoding human ACE2 was kindly provided by Thomas Gallagher (Loyola University). pcDNA3.1 encoding CoV-1 Spike or CoV-2 Spike tagged with a C9 epitope on the C-terminus, or MERS Spike, was kindly provided by Thomas Gallagher (Loyola University). pcDNA3.1 encoding CoV-1 Spike or CoV-2 Spike tagged with a FLAG epitope on the C-terminus was obtained from Michael Letko and Vincent Munster (NIAID). pMD2.G encoding VSV-G (12259) was obtained from Addgene (a generous gift from Didier Trono). pWPI was obtained from Addgene (12254) and human ACE2 or human TMPRSS2 was introduced by Gateway cloning (Gateway LR Clonase II Enzyme mix (11791020)) as per manufacturer’s instructions. pPolII encoding hemagglutinin (HA) or neuraminidase (NA) from Influenza A/Turkey/1/2005 (H5N1) were kindly provided by Richard Yi Tsun Kao (The University of Hong Kong). pCMV encoding HIV-1 Vpr fused to beta lactamase (pCMV4-BlaM-Vpr) was obtained from Addgene (21950).
Techniques: Incubation, Flow Cytometry, Infection, Luciferase, Activity Assay
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