Fig. 3: USP22 negatively regulates STAT1 signaling and IFN-λ1 expression.
From: USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING
A. Basal mRNA expression levels of total IFNA (panIFNA) and IFNB1 in control (NHT) and the CRISPR/Cas9-generated USP22 KO HT-29 single clone (USP22 KO #62). Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; **P < 0.01. B Western blot analysis of basal phosphorylated and total levels of STAT1 and USP22 in control and USP22 KO HT-29 cells (USP22 KO #62). GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. C FACS-based analysis of the indicated basal secretion patterns of the viral defense cytokine panel in supernatants of control and USP22 KO HT-29 cells (USP22 KO #62). Data are presented as absolute levels of cytokines (in pg/ml). Samples below lower detection limit were set to zero, values above upper detection limit were set to detection limit. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; n.s. not significant. D Basal mRNA expression levels of IFNL1 in control and USP22 KO HT-29 single clone (USP22 KO #62). Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05. E Western blot analysis of phosphorylated and total levels of STAT1 as well as USP22 in control and USP22 KO HT-29 cells (USP22 KO #62) after 24 h incubation with 1 µg/ml IFNAR2 blocking antibody. As positive control, cells were pre-treated for 1 h with IFNAR2 blocking antibody, then treated 1 h with 0.05 ng/ml IFN-β. Vinculin served as loading control. Representative blots of at least two different independent experiments are shown.
