SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

Shi, Chong-Shan; Nabar, Neel R.; Huang, Ning-Na; Kehrl, John H.

Myc tagged NLRP3 plasmids were kindly provided by Dr. Yong-Jun Liu (Baylor). The Myc tagged NLRP3 LRR domain construct spanned amino acids 537-991 in NLRP3. The pcDNA3 NLRP3-Flag construct was kindly provided by Dr. Gabriel Nunez (University of Michigan). To construct the NLRP3 Ds-Red construct, the Ds-Red coding region was PCRed from the pDs-Red-Monomer fluorescent vector (Clontech) and inserted into Flag tagged NLRP3 construct after removal of the c-terminal Flag coding sequence. The TFEB-GFP plasmid was bought from Addgene, (38119) and the TFEB-mCherry plasmid made by moving the TFEB cDNA into the mCherry N1 vector (ClonTech). The ptf-Galectin 3 plasmid was also purchased from Addgene (64149), and the mCherry-Galectin 3 plasmid made by removing the GFP from the ptf-Galectin 3 vector. Pooled siRNAs targeting NLRP3 (sc-45469), ASC (sc-37281), GFP (sc-45924), and a scrambled control (sc-37007) were purchased from Santa Cruz Biotechnology. Plasmids and siRNA were transfected into cells using X-tremeGENE-HP (Roche, 6366236001) following the manufacture's protocol.