Fig. 4: Role of free versus conjugated ISG15 on viral replication and cytokine secretion.
From: Altered ISGylation drives aberrant macrophage-dependent immune responses during SARS-CoV-2 infection
a, Macrophages were transfected with ISG15, UBE1L, HERC5 or USP18 DsiRNA for 72 h; depletion was verified in type I IFN-treated cells by immunoblotting. b, Cellular ISGylation was measured in Zika- and SARS-CoV-2-infected cells (MOI = 2), 24 h post-infection. a,b, Immunoblots are representative of three biologically independent experiments. c, Total RNA was collected at the indicated time intervals from Zika-infected cells; quantifications of absolute copy number were done by RT–qPCR using universal viral RNA-specific primers. Data are displayed as the mean ± s.e.m. of three biologically independent experiments. *P < 0.05 by Mann–Whitney U-test versus control. d, The indicated cytokines were quantified by cytometric beads assay from macrophages transfected with either NT, ISG15, UBE1L, HERC5 or USP18 DsiRNA for 72 h before infection. Free ISG15 was determined by ELISA. Data are displayed as the mean ± s.e.m. of three biologically independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Mann–Whitney U-test versus control. e, Total RNA was collected at the indicated time intervals from SARS-CoV-2-infected cells; quantifications of absolute copy number were done by RT–qPCR using viral RNA-specific primers. Data are displayed as the mean ± s.e.m. of three biologically independent experiments. *P < 0.05 by Mann–Whitney U-test versus control. f, The indicated cytokines were quantified by cytometric beads assay from macrophages transfected with either NT, ISG15, UBE1L, HERC5 or USP18 DsiRNA for 72 h before infection. Free ISG15 was determined by ELISA. Data are displayed as the mean ± s.e.m. of three biologically independent experiments. *P < 0.05 by two-tailed Mann–Whitney U-test versus control (NT cells). g, Macrophages were treated with ISG15 DsiRNA and infected with SARS-CoV-2 (MOI = 2) in media supplemented with exogenous purified ISG15 (1 µg ml−1); cells were either untreated or pretreated with anti-LFA-1 inhibitory antibody (500 ng) for 1 h before infection. The indicated cytokines were quantified by cytometric beads assay. The error bars represent the mean ± s.d. *P < 0.05, **P < 0.01 by two-tailed Mann–Whitney U-test versus control cells (n = 3 biologically independent experiments).
