Journal: bioRxiv

Article Title: SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike-ACE2 receptor interaction

doi: 10.1101/2022.01.11.475947

Figure Lengend Snippet: Microglia signature markers, P2RY12 and TMEM119 (in green) and cells nuclei (in blue) assessed by immunofluorescence staining, and representative western blot are presented in panel ( A-B ), respectively. Growth kinetics of SARS-CoV-2 (D614) on MDMi, mouse microglia (mMi), Vero E6 and Caco2 cells in ( C ). Relative expression of ACE2 in MDMi by qPCR compared to Vero E6 and Hek-293T in ( D ). Level of ACE2 receptor in MDMi and mouse microglia compared to Caco2 and Vero E6 cells analysed by western blot shown in panel ( E ). Viral RNA levels from SARS-CoV-2 particles bound on cell surface expressed as N2 copies/well in ( F ) Intracellular luciferase level (LUC) delivered by pseudo-virus (PV) particle for SARS-CoV-2 in MDMi and Vero E6 compared to the non-glycoprotein control (NE) in ( G ). SARS-CoV-2 replication on MDMi (at MOI of 1) and Vero E6 (at MOI of 0.01) using SARS-CoV-2 reporter virus expressing ZsGreen fluorescent protein assessed directly under confocal microscopy at 3dpi are shown in panel ( H ). Data points are means + SEM from at least three different donors. *P < 0.05, **P < 0.01, and ***P < 0.001 and **** P < 0.0001 by two-way ANOVA test with Sidak’s correction.

Article Snippet: The human ACE2 transcript variant 2 was amplified using the OriGene primer set Forward: 5’-TCC ATT GGT CTT CTG TCA CCC G-3’ and Reverse: 5’-AGA CCA TCC ACC TCC ACT TCT C-3’.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Luciferase, Confocal Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike-ACE2 receptor interaction

doi: 10.1101/2022.01.11.475947

Figure Lengend Snippet: Relative of infectivity determined by Plaque Reduction Neutralisation Test (PRNT) to verify the neutralizing level of a soluble receptor hACE2-FcM compared to a non-related SARS-CoV-2 receptor NCAM-FcM (top) and the inhibitory concentration (IC50) (bottom) ( A ). Spike–mediated IL-1β secretion (supernatant) in vehicle (untreated) or LPS-primed MDMIs exposed to S-clamp (S; 10-50 μg) in presence or absence of the soluble hACE2-FcM protein. ATP (5 mM) treatment for 1 hour was used as a positive control ( B ). Inhibition of spike-mediated IL-1β secretion by ACE2 inhibition with MLN-4760 (1 or 10 μM) ( C ). Validation of low endotoxin anti-ACE2 (3E8) and anti-Hemagglutinin from influenza A H3 (CO5) proteins by SDS-PAGE and ELISA ( D-E ). Effect of 3E8 in blocking cells activation by spike protein in pre-treatment of LPS-primed MDMi exposed to S-clamp ( F ). Data are means + SEM from at least three different donors. *P < 0.05, **P < 0.01, and ***P < 0.001 and **** P < 0.0001 by one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

Article Snippet: The human ACE2 transcript variant 2 was amplified using the OriGene primer set Forward: 5’-TCC ATT GGT CTT CTG TCA CCC G-3’ and Reverse: 5’-AGA CCA TCC ACC TCC ACT TCT C-3’.

Techniques: Infection, Concentration Assay, Positive Control, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay, Blocking Assay, Activation Assay