Journal: Cell Death & Disease

Article Title: SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death

doi: 10.1038/s41419-018-0917-y

Figure Lengend Snippet: Confocal imaging analysis of ( a ) HeLa cells co-transfected with Galectin3-mCherry and SARS 3a-GFP, to assay lysosomal damage. b Quantification of Galectin3-mCherry spots/cell from GFP-vector (GPFV) or SARS 3a-GFP (3a GFP) expressing cells ( n = 15 cells). c Quantification of colocalization (PCC) between Galectin3-mCherry and either GFP-vector or SARS 3a-GFP ( n = 15 cells). d HeLa cells transfected with SARS 3a-GFP and incubated with DQ-BSA red to assay lysosomal degradation capacity. ×100 electronic zoom, scale bar 10 μm. e Quantification of multiple images from d , data shown as normalized intensity. f Immunoblot of lysates from 293T cells expressing SARS 3a-flag and HA-bid with or without Z-VAD or E64D and pep A. The western data are representative of two or three independent experiments (*** p < 0.0001, unpaired Student’s T- test)

Article Snippet: Lamp1-mCherry plasmid and LAMP1-CFP plasmids were obtained from the Jenifer Lippincott-Shwartz lab; PPP3CB siRNA was bought from Santa Cruz (SC-39195); the NLRP3 flag plasmid was obtained from Gabriel Nunez’s Lab at the University of Michigan Med School; Caspase-1 flag plasmid, IL-1β-flag plasmid, and ASC-flag plasmids were from the lab of Katherine A Fitzgerald at the University of Massachusetts Medical School; TFEB-GFP was bought from Addgene (38119), and the TFEB-mCherry plasmid was made by moving the TFEB cDNA into the mCherry N1 vector (ClonTech); NEK7 shRNA was made using the targeting sequence: 5′-C​C​G​G​T​G​G​A​G​T​G​C​C​G​G​T​A​G​C​G​T​T​A​A​A​C​T​C​G​A​G​T​T​T​A​A​C​G​C​T​A​C​C​G​G​C​A​C​T​C​C​A​T​T​T​T​T​G​-3′ and inserting into the pSIREN vector (ClonTech); pRip3-mCherry(61386) and ptf-Galectin3 (64149) were purchased from Addgene, and mCherry-Galectin3 was made by removing the GFP from ptf-Galectin3.

Techniques: Imaging, Transfection, Plasmid Preparation, Expressing, Incubation, Western Blot