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IFNAR1 −/− mice (n=6-8) were pretreated with FFAR2 inhibitor 4-CMTB followed by PBA, NaB, or NaAc via i.p. administration, and ZIKV infection as per the timeline shown in . Seven days post-infection, anterior segment/TM tissue from treated and untreated mice were harvested and subjected to (A) RNA extraction and qPCR to measure the mRNA expression levels of PRRs (RIG-I, TLR3), inflammatory (IL-6, IL-1β, CCL-4), IFNs (IFN-α2, IFN-β1), and ISGs (ISG15, OAS2, MX1), and (B) western blot for NFκB, MAPKs (ERK1/2, p38), STAT1, <t>STAT3,</t> RIG-I, and IRF7 inflammatory/ISG pathways. (C) Densitometric analysis of the immunoblots was performed using ImageJ. The bar graph represents means ± SD from three biological replicates. * P < 0.05; ** P < 0.005; *** P < 0.0005; ****, P < 0.0001 (one-way ANOVA).
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IFNAR1 −/− mice (n=6-8) were pretreated with FFAR2 inhibitor 4-CMTB followed by PBA, NaB, or NaAc via i.p. administration, and ZIKV infection as per the timeline shown in . Seven days post-infection, anterior segment/TM tissue from treated and untreated mice were harvested and subjected to (A) RNA extraction and qPCR to measure the mRNA expression levels of PRRs (RIG-I, TLR3), inflammatory (IL-6, IL-1β, CCL-4), IFNs (IFN-α2, IFN-β1), and ISGs (ISG15, OAS2, MX1), and (B) western blot for NFκB, MAPKs (ERK1/2, p38), STAT1, STAT3, RIG-I, and IRF7 inflammatory/ISG pathways. (C) Densitometric analysis of the immunoblots was performed using ImageJ. The bar graph represents means ± SD from three biological replicates. * P < 0.05; ** P < 0.005; *** P < 0.0005; ****, P < 0.0001 (one-way ANOVA).

Journal: bioRxiv

Article Title: Gut microbial metabolites butyrate and acetate limit Zika virus replication and associated ocular manifestations via the G-protein coupled receptor 43/FFAR2

doi: 10.1101/2025.07.15.664962

Figure Lengend Snippet: IFNAR1 −/− mice (n=6-8) were pretreated with FFAR2 inhibitor 4-CMTB followed by PBA, NaB, or NaAc via i.p. administration, and ZIKV infection as per the timeline shown in . Seven days post-infection, anterior segment/TM tissue from treated and untreated mice were harvested and subjected to (A) RNA extraction and qPCR to measure the mRNA expression levels of PRRs (RIG-I, TLR3), inflammatory (IL-6, IL-1β, CCL-4), IFNs (IFN-α2, IFN-β1), and ISGs (ISG15, OAS2, MX1), and (B) western blot for NFκB, MAPKs (ERK1/2, p38), STAT1, STAT3, RIG-I, and IRF7 inflammatory/ISG pathways. (C) Densitometric analysis of the immunoblots was performed using ImageJ. The bar graph represents means ± SD from three biological replicates. * P < 0.05; ** P < 0.005; *** P < 0.0005; ****, P < 0.0001 (one-way ANOVA).

Article Snippet: Antibodies used in this study were purchased from the following sources: 4G2 (GeneTex, #GTX57154), NS3 (GeneTex, #GTX133309), β-actin (Millipore Sigma, #A2228), pNFκB (#3033), NFκB (#6956) pERK1/2 (#4370), ERK (#4695), pP38 (#4511), P38 (#8690), pSTAT1 (#9167), STAT1 (#14994), pSTAT2 (#88410), STAT2 (#72604), pSTAT3 (#9145), STAT3 (#9139), RIG-I (#3743), pIRF3 (#79945), IRF3 (#4302), and IFIT2 (#92633) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Infection, RNA Extraction, Expressing, Western Blot