Fig 2: Schema of workflow of PLA developed for detecting target-specific interactions between RNA sequences and RBP(A) RNA probe hybridization was performed using a biotinylated PLA probe (black with biotin indicated in red), which targets a specific mRNA sequence in MYC gene close to the binding site of FXR1 protein (yellow) within MYC mRNA (purple line with FAM indicated in green at the 5′end) is performed.(B) Two primary antibodies raised in different species are added. In this case a rabbit antibody (blue) targets the FXR1, and a mouse antibody (red) targets the biotin label in the PLA probe are used.(C) Two secondary PLA species-specific antibodies (green and pink) conjugated with oligonucleotides are then added.(D) The conjugated oligonucleotides are joined and circularized by ligation upon introduction of linear connector oligonucleotide (black).(E) The PLA probe acts as a primer for target mRNA generated concatemeric sequences during rolling circle replication (the amplified circular molecule is annotated in brown).(F) In the last step, labeled oligos hybridized to the complementary sequences within the amplicon are visualized and quantified as discrete spots of PLA signals using microscopy.

Fig 3: xrRNA1 is required and sufficient for FMRP binding to the ZIKV 3' UTR.(A) Schematic illustrating the secondary structures present in the ZIKV 3' UTR and RNAs used for affinity chromatography. Deletion mutant RNA constructs fused to a tobramycin aptamer at the 5' end are shown. DENV NS2A coding sequence was used as a negative control RNA. (B) Purified RNAs bound to tobramycin-sepharose beads were incubated with HeLa cell lysate and unbound proteins were washed away prior to elution for western blotting for DDX6, FMRP, FXR1, FXR2, G3BP1 and PTB.

Fig 4: FXR-nsP3 complexes bind viral G RNA in VEEV-infected cells.(A) Colocalization of VEEV nsP3-GFP fusion proteins with VEEV G RNA. Images are presented as MIP of 6 x-y sections (1 μm) through the middle plane on nucleus. Bars: 10 and 3 μm. (B) Colocalization of FXR1-GFP with VEEV G RNA. Images are presented as MIP of 6 x-y sections (1 μm) through the middle plane of the nucleus. Bars: 10 and 3 μm. (C) Localization of VEEV nsP3-GFP, FXR1 and dsRNA at the plasma membrane at 2 h PI. Images are presented as MIP of x-z sections (1 μm). Bars: 10 μm. (D) The x-y 1-μm-thick section at the plasma membrane of the cell presented in C. The dsRNAs are presented as spots rendered in Imaris software. Bars: 3 and 1 μm. (E) Localization of VEEV nsP3-GFP, dsRNA and G RNA at the plasma membrane at 2 h PI. Images are presented as MIP of x-y 1-μm-thick sections at the plasma membrane. The dsRNAs are presented as spots rendered in Imaris. Bars: 10 and 3 μm. Yellow arrowheads indicate colocalization of nsP3-GFP and G RNA in A or nsP3-GFP and FXR1 in D. Red arrowheads indicate G RNA spots lacking nsP3-GFP. Green arrowheads indicate positions of nsP3-GFP complexes, containing no G RNA. Turquoise arrowheads indicate fully formed, dsRNA-positive vRCs, which contain low levels of G3BP1-GFP. Pink arrowheads indicate the dsRNA signals of low intensity and volume, which is suggesting the presence of shorter dsRNAs.

Fig 5: Effects of emodin on molecules of BSEP pathway in rat primary hepatocytes. Immunofluorescence was used to detect CK-18 protein in hepatocytes, and the red fluorescence molecule Dylight488 in primary hepatocytes was observed under a fluorescence microscope (A–C). Cytotoxicity of emodin on primary hepatocytes was determined by CCK8 assay (D). The mRNA levels of FXR and BSEP were detected by qRT-PCR (E). The protein levels of FXR1, FXR2, and BSEP were detected by Western blotting (F,G). Data are shown as the mean ± SD. n = 5. (∗P < 0.05 compared to the control group; ∗∗P < 0.01 compared to the control group).