Fig. 3: Viperin regulates SGK1 expression by lowering STAT1 levels.
From: Targeting Viperin prevents coxsackievirus B3-induced acute heart failure
a Whole cell lysates from HEK293T cells transfected with Flag-Viperin were subjected to immunoprecipitation with Flag agarose (left). Mass spectrometry analysis of potential Viperin-binding TFs was shown (right). The predicted TFs of SGK1 were from the KnockTF2.0 database. b RT-qPCR analysis of Sgk1 mRNA in HEK293T cells (WT or knockout of the indicated TFs) transfected with Flag-Viperin. c RT-qPCR analysis of Sgk1 mRNA in HL-1 cells transfected with control shRNAs (shCtrl) or shRNAs against STAT1 (shSTAT1), together with or without Flag-Viperin. d RT-qPCR analysis of Sgk1 mRNA in HL-1 cells transfected with increasing amounts of Flag-STAT1. e Western blot analysis of SGK1 levels in HL-1 cells transfected with shCtrl (‒) or shSTAT1 (#1 and #2). f Confocal microscopy in HeLa cells transfected with Flag-Viperin and eGFP-STAT1. Scale bar, 5 μm. g Immunoprecipitation analysis of the interaction between Viperin and STAT1 in HL-1 cells infected with CVB3 (MOI = 10) for 12 h. h GST pulldown assay was performed to analyze the interaction between Viperin and STAT1. i Western blot analysis of STAT1 levels in HL-1 cells transfected with increasing amounts of HA-Viperin. j Western blot analysis of SGK1 levels in HL-1 cells transfected with shCtrl or shSTAT1, together with or without Flag-Viperin. k Western blot analysis of SGK1 levels in HL-1 cells transfected with shCtrl or shSTAT1, followed by CVB3 infection (MOI = 10) for 12 h. l Western blot analysis of SGK1 and Viperin levels in Stat1+/+ or Stat1−/− cells infected with or without CVB3 (MOI = 10). m Mice were injected the siCtrl or siSTAT1 in the tail vein at a dose of 0.1 mg per mouse once a day for four consecutive days, and then were infected with or without CVB3 (2 × 105 PFU per gram body, i.p.) for 7 days. Western blot analysis was used to analyze SGK1, STAT1 and cytomembrane KCNQ1 levels. NS not significant (P > 0.05), ***P < 0.001, two-tailed unpaired Student’s t-test (b, c), one-way analysis of variance (ANOVA) (d). Data are shown as means ± SD of four biological replicates (b‒d) or are representative of three independent experiments (e, g‒m).
