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. 2021 Aug 26;18(4):891–908. doi: 10.1080/15548627.2021.1963155

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Figure 7. — USP19 deficiency in macrophages enhanced VSV-induced type I interferon production and antiviral response by suppressing CMA-mediated TBK1 degradation. (A and B) qPCR analysis of Ifna4 (A) and Ifnb1 (B) expression in Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated times with or without VER-155,008 (5 μM) pretreatment. (C and D) qPCR analysis of Ifna4 (C) and Ifnb1 (D) expression in Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages transfected with poly(I:C) (1 μg/mL) for the indicated hours with or without pretreatment with VER-155,008 (5 μM). (E and F) qPCR analysis of Ccl5, Cxcl10 (E), Isg15, Ifit2, Ifit1, and Mx1 expression (F) in Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for 12 h with or without pretreatment with VER-155,008 (5 μM). (G) Immunoblot analysis of OSA1, EIF2AK2, and LAMP2A expression in Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated durations. (H and I) Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages were infected with VSV-GFP for 12 h with or without VER-155,008 (5 μM) pretreatment, and images were captured under a fluorescence microscope (H). The percentage of GFP⁺ cells was determined via flow cytometry (I). (J) qPCR analysis of VSV replication in Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) with or without VER-155,008 (5 μM) pretreatment. (K) Determination of VSV titers in supernatants via a TCID₅₀ assay of Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages infected with VSV with or without VER-155,008 (5 μM) treatment. (L) Immunoblot analysis of the indicated proteins in Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated durations. (M) Immunoblot analysis of STAT1 and STAT2 phosphorylation in Usp19^flox/flox and usp19^flox/flox Lyz2-Cre peritoneal macrophages infected with VSV (MOI = 1) for the indicated hours. The data are representative of three independent experiments. Scale bars: 100 μm. Error bars show the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t test.