Journal: eLife

Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

doi: 10.7554/elife.41159

Figure Lengend Snippet: Figure 3. miR-122 enhances IFN response by suppressing p-STAT3. (A) Luciferase activity of a STAT3-responsible promoter construct in HepG2 cells co-transfected with mimics (NC or miR-122) for 2 days, and then transfected with SGR-JFH1 RNA for the indicated time. (B) Western blot analysis of total and phosphorylated STAT3 (p-STAT3, Tyr705) in HepG2 cells first transfected with mimics (NC or miR-122) for 2 days, and then treated with JFH1 RNA or poly(I:C). (C) Analysis of STAT3 protein in HepG2 cells treated with mimics (NC or miR-122) or siRNAs (NC or STAT3). (D, E) Analysis of the mRNAs (D) and proteins (E) of IFNs in HepG2 cells treated with siRNAs (NC or STAT3) and then JFH1 RNA. Cells treated with miR-122 or NC mimics were used as controls in panel D. (F) Analysis of IFN mRNAs in HepG2 cells treated with siRNAs and then with poly(I:C). (G, H) Analysis of p-STAT1 protein and IFN mRNAs in HepG2 cells treated with either S3I-201 or cryptotanshinone (CST) for 24 hr, and then transfected with poly(I:C). Luciferase data are from three experiments (mean +SD). ELISA data are from two experiments (mean +SD). qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p < 0.05, **p < 0.01 and ***p < 0.001. DOI: https://doi.org/10.7554/eLife.41159.018 The following source data and figure supplements are available for figure 3:

Article Snippet: Recombinant cytokines (IL-29, 1598-IL-025; IFN-b, 8499-IF-010; IL-6, 206-IL-010) and ELISA kits (IL29/IL-28B, DY1598B; IFN-b, DY814) were obtained from R&D systems.

Techniques: Luciferase, Activity Assay, Construct, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: Production of IFN-α/β, IFN-λ, and TNF-α after Stimulation with Viruses in IRAK-4-Deficient Blood Cells PBMCs from controls and/or from IRAK-4-deficient patients P2, P3, or P7 were left unstimulated or stimulated with various intact and UV-inactivated viruses. (A) IFN-β mRNA levels were analyzed by reverse transcription and real-time quantitative PCR 24 hr after stimulation with intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for the controls from three independent individuals, each tested once. The patients were each tested once. (B and C) IFN-α secretion (B) and IFN-λ secretion (C) were measured by ELISA after 24 hr of stimulation. Means and standard deviations (SD) were calculated for the controls from 14 independent individuals, each tested once. (D) IFN-α secretion by control PBMCs was measured by ELISA after 24 hr of stimulation by intact or UV-inactivated viruses. Means and standard deviations (SD) were calculated for the controls from seven independent individuals, each tested once. (E) TNF-α secretion was measured by ELISA after 24 hr of stimulation by two intact viruses. Means and standard deviations (SD) for controls were calculated from two independent individuals.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: IFN-β, IFN-λ, and IL-6 Induction in IRAK-4-Deficient Fibroblasts upon Stimulation by Poly(I:C), IL-1β, TNF-α, and Viruses Induction of IFN-β, IFN-λ, and IL-6 in response to poly(I:C) (50 μg/ml), IL-1β (20 ng/ml), TNF-α (10 ng/ml), and viral stimulations in IRAK-4-deficient (P1, P2, or P4) and control fibroblasts. (A and B) IFN-β mRNA induction, as determined by Q-PCR 2 hr after stimulation (A) and IFN-β, as determined by ELISA 24 hr after stimulation (B). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (C) IFN-β and IFN-λ as determined by ELISA 24 hr after stimulation by poly(I:C), following prior treatment with IFN-α2b (10 5 U/ml for 12 hr). This experiment is representative of two independent experiments. (D) IL-6, as determined by ELISA 24 hr after stimulation. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (E) IFN-β mRNA induction 24 hr after stimulation by seven intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Infection