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. 2023 May 9;220(8):e20220727. doi: 10.1084/jem.20220727

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© 2023 Lin et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S3. — ZNRF1 deficiency in MEFs and BMDMs enhances IFN production and restricts EMCV proliferation after viral infection. (A–D) Znrf1^+/+ and Znrf1^−/− MEFs were infected with EMCV at MOI of 1 for the times indicated. (A) RT-qPCR analysis of the mRNA expression of the genes indicated. (B) Immunoblot analysis of p-IKKα/β, p-IRF3, and phosphorylation of MAPKs in Znrf1^+/+ and Znrf1^−/− MEFs. (C) The viral titers in culture media were determined by plaque assays. (D) Quantification of viral particles in C. (E and F) Znrf1^+/+ and Znrf1^−/− BMDMs were infected with EMCV at an MOI of 5 for the times indicated. (E) RT-qPCR analysis of the expression of EMCV 2A2B RNA in BMDMs. (F) Quantification of viral particles in culture media was made by plaque assays. (G) Sequence analysis of wild-type and two different ZNRF1^−/− Calu-3 clones generated by the CRISPR/Cas9 system. Genomic DNA was extracted from wild-type and ZNRF1^−/− Calu-3 cells and the region surrounding the targeted site was amplified by PCR for sequencing. Indel mutations are indicated in red. (H) Immunoblot analysis of ZNRF1 protein in cell lysates from scrambled controls (sgCtrl) and ZNRF1^−/− Calu-3 and RAW264.7 cells. (I) Calu-3 cells treated with poly(I:C) (30 μg/ml) or R848 (2 μM) for 4 h. RT-qPCR analysis of the expression of IFNB in Calu-3 cells. (J) The RNA level of Znrf1 in patients with mild-to-moderate (n = 10), severe (n = 10), and critical (n = 10) COVID-19 were normalized with healthy controls for clinical validation. Data were analyzed from the National Center for Biotechnology Information GEO database, accession number: GSE167930. (K) Immunoblot analysis of ZNRF1 protein in lysates from Znrf1^−/− iBMDMs reconstituted with vector, wild-type ZNRF1, or ZNRF1(C184A) mutant. (L) The secretion of IFN-β, IL-6, and IL-10 into the culture media of Znrf1^−/− RAW264.7 cells reconstituted with vector, wild-type ZNRF1, ZNRF1(C184A), or ZNRF1(Y103F) mutants after stimulation with poly(I:C) (30 μg/ml) for 4 h were measured by ELISA. (M and N) HEK293T were co-transfected with IFN-β-Luc (M) or NF-κB-Luc (N) reporter, wild-type ZNRF1 or ZNRF1(C184A) mutant, and the pattern-recognition receptors indicated for 36 h. Cells were harvested, and reporter activities were analyzed by the dual-luciferase reporter assay. The expression of the proteins indicated in cell lysates was confirmed by immunoblotting, as shown in the lower panel. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test). Data are representative of three independent experiments (error bars, mean ± SD). Source data are available for this figure: SourceData FS3.