Journal: Scientific Reports

Article Title: TRBP modulates RLR signaling by inhibiting PKR-mediated antiviral stress granule formation

doi: 10.1038/s41598-025-07121-3

Figure Lengend Snippet: TRBP negatively regulates RLR-mediated IFN signal. (a–c) L929 cells were transfected with the IFN reporter gene (p-125Luc) and internal control (pRL-tk) vectors, together with the expression vector for Empty or TRBP. After 48 h transfection, the cells were mock-treated (mock) or stimulated by IAVΔNS1 infection (a) , polyIC transfection (b) , and SenV infection (c) for 12 h and subjected to a dual-luciferase assay. (d) HeLa WT and TRBP KO cells (#1, #2, #3) were mock-treated (mock) or transfected with polyIC for 9 h, and IFN-β mRNA expression levels were determined by qRT-PCR. (e) HeLa WT, TRBP KO (#2, #3), and FLAG-TRBP- expressing TRBP KO cells were infected with IAVΔNS1. After 18 h incubation, cell extracts were prepared and subjected to SDS-PAGE, and immunoblotted using antibodies against phospho-IRF-3 (Ser386), IRF-3, TRBP, FLAG-tag, IAV NP, and β-actin (ACTB). The intensities of the p-IRF-3 bands were normalized against ACTB, measured using ImageJ software. The data represent the mean results of three independent experiments. (f) Flp-In 293 CBP-SBP-GFP (GFP) or FLAG-TRBP (TRBP) cells were treated with doxycycline (Dox) 0 or 1.0 µg/mL for 16 h. The cells were infected with IAVΔNS1 for 9 h, and IFN-β mRNA level was determined by qRT-PCR. (g) Flp-In 293 CBP-SBP-GFP (GFP) (left) or FLAG-TRBP (FLAG) (right) cells were treated with DOX for 16 h and then the cells were mock-transfected or transfected with short or long polyIC for 9 h. IFN-β mRNA expression levels were determined by qRT-PCR. At least two independent experiments represent similar results, and the bars indicate one representative; each bar shows the means and ± the standard deviation (S.D.) of the technical triplicate. P-values were determined by Student’s t-test (* P < 0.05).

Article Snippet: Polyinosinic-polycytidylic acid (polyIC) was purchased from Amersham (UK).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Infection, Luciferase, Quantitative RT-PCR, Incubation, SDS Page, FLAG-tag, Software, Standard Deviation

Journal: Scientific Reports

Article Title: TRBP modulates RLR signaling by inhibiting PKR-mediated antiviral stress granule formation

doi: 10.1038/s41598-025-07121-3

Figure Lengend Snippet: TRBP inhibits PKR activation and avSG formation. (a) Flp-In 293 CBP-SBP-GFP (GFP) or FLAG-TRBP cells were treated with doxycycline (Dox) 0 or 1.0 µg/mL for 14 h. The cells were mock-treated (mock), infected with NDV or IAVΔNS1, or transfected with polyIC. After 12 h incubation, cell extracts were prepared and subjected to SDS-PAGE, and immunoblotted using antibodies against GFP, FLAG-tag, PKR, phospho-PKR (Thr 446), eIF2α, phospho-eIF2α (Ser 51), NDV NP, IAV NP, and ACTB. The intensity of the p-PKR and p-eIF2α bands was normalized against ACTB, measured using ImageJ software. (b) Flp-In 293 FLAG-TRBP cells were treated with DOX 0 or 1.0 µg/mL for 24 h, and infected with IAVΔNS1 for 12 h. The cells were stained with anti-FLAG (Green = TRBP), anti-IAV NP (Red), anti-TIAR (Gray) antibodies, and DAPI (Blue). The white scale bar corresponds to 10 μm. The percentages of SG-containing cells were calculated in more than 10 randomly chosen fields for each slide ( n > 660 cells). (c) Flp-In 293 FLAG-TRBP cells were treated with DOX 0 or 1.0 µg/mL for 24 h, and treated with NaAsO 2 (0.5 mM) for 30 min and stained with anti-FLAG (Green = TRBP), anti-TIAR (Red) antibodies and DAPI (Blue). The white scale bar corresponds to 10 μm. The percentages of SG-containing cells were calculated in more than six randomly chosen fields for each slide ( n > 280 cells). (d) Flp-In 293 CBP-SBP-GFP (GFP) or FLAG-TRBP (TRBP) cells were transiently transfected with p-125Luc and pRL-tk reporter vectors, together with the RIG-I CARD, MAVS, and IRF-3-5D expressing vectors, respectively. After 24 h transfection, the cells were treated with DOX 0 (-) or 1.0 (+) µg/mL for 24 h and subjected to a dual-luciferase assay. Luciferase activity is normalized against each DOX (-) as 100%. At least two independent experiments represent similar results, and the bars indicate the one representative; each bar shows the means and ± the standard deviation (S.D.) of the technical triplicate. P-values were determined by Student’s t-test (* P < 0.05).

Article Snippet: Polyinosinic-polycytidylic acid (polyIC) was purchased from Amersham (UK).

Techniques: Activation Assay, Infection, Transfection, Incubation, SDS Page, FLAG-tag, Software, Staining, Expressing, Luciferase, Activity Assay, Standard Deviation

Journal: Scientific Reports

Article Title: TRBP modulates RLR signaling by inhibiting PKR-mediated antiviral stress granule formation

doi: 10.1038/s41598-025-07121-3

Figure Lengend Snippet: dsRBDs of TRBP are essential to regulate avSG-mediated IFN signal. (a) L929 cells were transfected with p-125Luc and pRL-tk reporter vectors, together with the empty (-), TRBP WT, or TRBP dsmut1 + 2 (0.25, 0.625, or 1.25 µg) expression vectors. The cells were mock-treated or infected with polyIC for 12 h and subjected to a dual-luciferase assay. Data represent relative firefly luciferase activity, normalized to Renilla luciferase activity. (b) HEK293T cells were transfected with Myc-tagged TRBP (TRBP-Myc) WT or dsmut 1 + 2. The cells were mock-treated or infected with IAVΔNS1 for 18 h. Cell extracts were prepared and subjected to SDS-PAGE, and immunoblotted using antibodies against phospho-PKR (Thr 446), PKR, IAV NP, Myc-tag, and ACTB. The intensities of the p-PKR bands were normalized against ACTB, measured using ImageJ software. The data represent the means of three independent experiments. (c) L929 cells were transfected with p-125Luc and pRL-tk reporter vectors, together with the empty, TRBP WT, D234A, or 1-234 expression vectors. The cells were mock-treated or infected with IAVΔNS1 (left) or transfected with polyIC (right) for 12 h and subjected to a dual-luciferase assay. Data represent relative firefly luciferase activity, normalized to Renilla luciferase activity. (d) L929 cells were transfected with p-125Luc and pRL-tk reporter vector, together with the empty, TRBP WT, S4A, or S4D (0.25 or 1.25 µg) expression vectors. The cells were mock-treated or infected with IAVΔNS1 for 12 h and subjected to a dual-luciferase assay. Data represent relative firefly luciferase activity, normalized to Renilla luciferase activity. At least two independent experiments represent similar results, and the bars indicate one representative; each bar shows the means and ± the standard deviation (S.D.) of the technical triplicate. P-values were determined by Student’s t-test (* P < 0.05).

Article Snippet: Polyinosinic-polycytidylic acid (polyIC) was purchased from Amersham (UK).

Techniques: Transfection, Expressing, Infection, Luciferase, Activity Assay, SDS Page, Software, Plasmid Preparation, Standard Deviation

Journal: Scientific Reports

Article Title: TRBP modulates RLR signaling by inhibiting PKR-mediated antiviral stress granule formation

doi: 10.1038/s41598-025-07121-3

Figure Lengend Snippet: LGP2 and PACT are not involved in the interaction between TRBP and PKR. (a) HEK293T cells were transfected with empty vector, Myc-tagged TRBP WT, or HA-tagged PKR K296R together with empty vector, FLAG-LGP2, FLAG-PACT, or both. After 48 h incubation, cell extracts were subjected to IP with anti-Myc-antibody. Input and IP samples were subjected to SDS-PAGE and immunoblotted using antibodies against HA-tag, Myc-tag, and FLAG-tag. (b) WT, PKR KO, or LGP2 KO MEF cells were transfected with p-125Luc and pRL-tk reporter vectors, together with the empty or TRBP WT expression vector. The cells were transfected with polyIC for 12 h and subjected to a dual-luciferase assay. Data represent luciferase activity normalized against MEF transfected with empty vector followed by polyIC transfection as 100%. At least two independent experiments represent similar results, and the bars indicate one representative; each bar shows the means and ± the standard deviation (S.D.) of the technical triplicate. P-values were determined by Student’s t-test (* P < 0.05).

Article Snippet: Polyinosinic-polycytidylic acid (polyIC) was purchased from Amersham (UK).

Techniques: Transfection, Plasmid Preparation, Incubation, SDS Page, FLAG-tag, Expressing, Luciferase, Activity Assay, Standard Deviation