Journal: Frontiers in microbiology

Article Title: Paracrine IFN Response Limits ZIKV Infection in Human Sertoli Cells.

doi: 10.3389/fmicb.2021.667146

Figure Lengend Snippet: FIGURE 2 | IFN response limits ZIKV infection in Sertoli cells. SC were exposed to ZIKV for 1h (MOI 3) and ZIKV infection and MX1 protein levels were evaluated by (A) immunofluorescence assay (IFA) using mouse monoclonal against the ZIKV E protein (green) and rabbit polyclonal against MX1 (red), respectively. Nuclei were stained with DAPI (blue). Images were taken at 100× magnification. (B) ZIKV titers in SC supernatant were (black line curve) determined by plaque assay (n = 3 for each time point) and the percentage of ZIKV-positive cells (green line curve) was quantified via IFA (n = 2 field per coverslip for each time point). (C) Changes in MX1 protein levels were quantified by mean fluorescence intensity (MFI) of IFA coverslips (n = 4) from each time point using ImageJ software, reported as MFI fold-change compared to mock. (D) Western blot analysis of MX1 protein levels at 24 and 48 h post-infection. b-actin and IRF3 were used as loading controls and each lane represents an independent experiment. (E) Secreted IFN-β in SC supernatant (n = 4 for each time point), determined by human IFN-β ELISA (R&D Systems); dotted line denotes detection limit of assay. (F) Zoomed images of IFA to depict co-localization of ZIKV and MX1 at 120h post-infection. (G,H) SC were treated with 5 pg/mL (1 IU/mL) of recombinant human IFN-β (rhIFN-β; R&D systems) 24 h prior to and upon ZIKV infection (MOI 1) and were replenished with the rhIFN-β (5 pg/mL) at 24 h post-infection. (G) ZIKV genome copies were measured in infected SC with and without IFN-β treatment at 48 h post-infection by RT-qPCR. (H) Gene expression of MX1 and IFIT1 was evaluated at 48 h post-infection by RT-qPCR in both mock and infected SC with and without IFN-β treatment and reported as fold-change compared to mock untreated. The housekeeping gene GAPDH was used to normalize fold-change for all gene expression assays. Significance determined by Student’s t-test for all assays, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Images were taken at 100× magnification. (B) ZIKV titers in SC supernatant were (black line curve) determined by plaque assay (n = 3 for each time point) and the percentage of ZIKV-positive cells (green line curve) was quantified via IFA (n = 2 field per coverslip for each time point). (C) Changes in MX1 protein levels were quantified by mean fluorescence intensity (MFI) of IFA coverslips (n = 4) from each time point using ImageJ software, reported as MFI fold-change compared to mock. (D) Western blot analysis of MX1 protein levels at 24 and 48 h post-infection. b-actin and IRF3 were used as loading controls and each lane represents an independent experiment. (E) Secreted IFN-β in SC supernatant (n = 4 for each time point), determined by human IFN-β ELISA (R&D Systems); dotted line denotes detection limit of assay. (F) Zoomed images of IFA to depict co-localization of ZIKV and MX1 at 120h post-infection. (G,H) SC were treated with 5 pg/mL (1 IU/mL) of recombinant human IFN-β (rhIFN-β; R&D systems) 24 h prior to and upon ZIKV infection (MOI 1) and were replenished with the rhIFN-β (5 pg/mL) at 24 h post-infection. (G) ZIKV genome copies were measured in infected SC with and without IFN-β treatment at 48 h post-infection by RT-qPCR. (H) Gene expression of MX1 and IFIT1 was evaluated at 48 h post-infection by RT-qPCR in both mock and infected SC with and without IFN-β treatment and reported as fold-change compared to mock untreated.

Techniques: Infection, Staining, Plaque Assay, Software, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Quantitative RT-PCR, Gene Expression