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. 2020 Sep 24;219(10):e201912074. doi: 10.1083/jcb.201912074

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© 2020 Wozniak et al.

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Figure S1. — Characterization of Inflammasome-induced exosome secretion. (A) EV release seen during HCV infection correlates with the appearance of a RILP cleavage product, as noted by Western blot. (B) Nanosight analysis of EVs isolated from virus-infected cells have a size range of 40–150 nm, consistent with that of exosomes. (C) EV protein content assessed by Bradford dye directly correlates with the total number of EVs as determined by NTA (Nanosight). Data are shown as mean ± SD; *, P ≤ 0.05 for n = 4–6. (D) EVs isolated from HCV-infected Huh-7.5 cells can be characterized by the exosomal markers CD81, flotillin, Rab7, and Rab27. They do not contain markers for microvesicles (annexin A1). WCL, whole-cell lysate. All markers were undetected in exosome-depleted medium. (E and F) HCV infection of Huh-7.5 cells increased expression of NLRP3, as noted by Western blot and pPCR. Cont., control. (G) Western blot analysis of Huh-7.5 cells expressing ncRILP-Flag with or without NLRP3-GFP showing equal protein expression. (H) shRNA to NLRP3 blocks HCV-induced exosome secretion. TRC, empty vector control. (I) LPS/ATP treatment of THP-1 cells does not up-regulate or activate caspase-4. The caspase-4 inhibitor Ac-LEVD-CHO was used at 100 µM. Ut, untreated. (J) Western blot analysis showing knockdown levels of NLRP3 and gasdermin D (GSDMD) in THP-1 cells.