Lipo293 transfection reagent

Microglia were transfected with lentivirus shRNA‐negative control (LV‐NC), lentivirus shRNA‐ACOD1 (LV‐ACOD1i), or lentivirus ACOD1 (LV‐ACOD1) at 10⁷ TU/mL supplemented 1 × HitransG A (GeneChem, Shanghai, China) for 12 h. The medium was then exchanged and the cells cultured for a further 60 h. Meanwhile, HEK‐293T cells were transfected with plasmid loading siRNA‐negative control (si‐NC), siRNA‐ACOD1 (si‐ACOD1) and ubiquitin‐Flag, Nrf2‐6his, ubiquitin‐K63‐HA, and ubiquitin‐K48‐MYC (GeneChem) at 1.5 μg/mL with Lipo293 Transfection Reagent (C0521, Beyotime) for 48 h. BV2 microglia were transfected with plasmid loading p‐p62 or mutation of p‐p62 (both from GeneChem) at 3 mg with the above reagent for 48 h.

The plasmids PNL4-3 luc R-E (designed to carry the pseudovirus skeleton protein-coded gene and luciferase reporter) and pVAX-Spike (a virus membrane protein-expressing plasmid) were selected using an E.Z.N.A.^® Endo-Free Plasmid DNA Maxi Kit (D6926). PNL4-3 luc R-E (8 μg) and pVAX-Spike (4 μg) were co-transfected into 293T cells using Lipo293™ transfection reagent c0521 (Beyotime Biotech, China) to generate the pseudovirus. The 293T-ACE2 cells were seeded in a 96-well plate (8,000 cells/well) overnight, and then the pseudovirus (100 μl) was seeded in DMEM (100 μl) for 1 h to prepare the mixture. Subsequently, the mixture was cocultured with 293T-ACE2 cells for 48 h. The pseudovirus-infected cells were detected using a Luciferase Reporter Gene Assay Kit 11401ES60 (Yeasen Biotech, China). The DMEM was removed, and the cells were washed with PBS. The cell lysate buffer (30 μl) was added to the Eppendorf tube, which was vortexed for 15 min at room temperature and spinned down (10,000 rpm, 15 min). The supernatant was removed to a new Eppendorf tube and mixed with Luciferase Reporter Gene Assay reagents (100 μl). The relative light unit (RLU) of the mixture was detected; the inhibition ratio of the pAbs was calculated by comparing the RLU of the pseudovirus-infected cell lysate with that of the mock control of cell lysate.

Lipo293 Transfection Reagent (Beyotime Biotechnology, C0521, China), dual-luciferase assay kits (Promega, E1910, USA). Flag-tag antibody (Sigma, F1804, USA), HA-tag antibody (Cell Signaling Technology, #2367, USA), and GAPDH antibody (Proteintech, 60004-1-Ig, China). HEK293T cells (CRL-3216) were purchased from ATCC (USA). DENV2 New Guinea C (NGC) strain was a gift from Prof. Tongyan Zhao (Beijing Institute of Microbiology and Epidemiology, China).