Journal: The Journal of Experimental Medicine

Article Title: Interferon-λ drives renal fibrosis by coordinating epithelial–fibroblast crosstalk

doi: 10.1084/jem.20251858

Figure Lengend Snippet: Type I and II IFNs differ from type III IFN in their regulation of TGF-β expression and the ERK–JNK pathway in renal fibroblasts during kidney fibrosis. (A–C) Primary renal fibroblasts from WT mice were treated for 24 h with 100 ng/ml of various IFNs subtypes (IFN-α, IFN-β, IFN-γ, or IFN-λ2) or PBS. (A and B) Isg15 , Mx1 , Ifit1 , and Tgf-β mRNA levels were assessed by RT-qPCR ( n = 6), and (C) TGF-β protein in the culture supernatants was quantified by ELISA ( n = 4). (D–F) Primary skin fibroblasts from WT mice were treated for 24 h with 100 ng/ml of various IFNs subtypes (IFN-α, IFN-β, IFN-γ, or IFN-λ2) or PBS. (D and E) Isg15 , Mx1 , Ifit1 , and Tgf-β mRNA levels were detected by RT-qPCR ( n = 6), and (F) TGF-β protein in the culture supernatants was measured by ELISA ( n = 4). (G–J) WT, Ifnar –/– , and Ifngr1 −/− mice were subjected to sham or UUO surgery, and kidneys were collected on day 7. n = 6 per group. (G) Representative images and quantitative analysis of fibrotic areas with Masson’s trichrome and PSR staining (scale bars = 50 μm). (H) RT-qPCR analysis of Acta2 , fibronectin, and vimentin mRNA levels in kidneys. TGF-β mRNA and protein levels in kidneys were measured by RT-qPCR (I) and western blot (J). (K and L) Primary kidney fibroblasts were treated with 100 ng/ml IFN-α (K) or IFN-β (L) for the indicated times. (K and L) Western blot analysis of phosphorylated and total ERK and JNK protein levels. Data in A–I are pooled from two independent experiments. Data in J–L are representative of three independent experiments. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparison test (A–I). ns, no significant difference. Source data are available for this figure: .

Article Snippet: Primary renal fibroblasts isolated from WT and Ifnlr1 −/− mice were stimulated with or without 100 ng/ml IFN-λ2 (250-33; PeproTech) for 1 h or 24 h. In separate experiments, primary renal and skin fibroblasts from WT mice were treated with 100 ng/ml of IFN-λ2 (250-33; PeproTech), IFN-α (CK83; Novoprotein), IFN-β (HY- P73130 ; MedChemExpress), or IFN-γ (315-05; PeproTech) for different times.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Comparison

Journal: Smart Molecules

Article Title: Endocytosis‐independent cytosolic entry of messenger RNA via fluorous bilayer zippering attenuating Toll‐like receptor signaling and enables ischemic tissue salvage

doi: 10.1002/smo2.70085

Figure Lengend Snippet: Biocompatibilities and immunogenicity of mRNA‐encapsulating polyplexes. (a) Cell viabilities of HUVECs upon 24 h incubation in presence of a variety of mRNA‐encapsulating polyplexes (mean ± s.d., n = 4, ** p < 0.01, student t test). (b) Hemolytic activities of sheep red blood cells upon 2 h incubation with varied concentrated delivery materials (*** p < 0.005; Student's t ‐test, mean ± s.d., n = 4). (c) confocal laser scanning microscopy (CLSM) measurement for assessment of overall cellular internalization of mRNA‐encapsulating polyplexes into RAW264.7 cells. (d) Interferon‐β (IFN‐β): at 4 h post incubation, the expression levels of inflammatory molecules were measured with qRT‐PCR. (e) Interleukin‐8 (IL‐8): at 4 h post incubation, the expression levels of inflammatory molecules were measured with qRT‐PCR. (** p < 0.01, *** p < 0.005; Student's t ‐test, n = 4).

Article Snippet: Culture supernatants (100 μL/well) were harvested and assayed for IFN‐α using the Mouse IFN‐α ELISA Kit (PBL Interferon Source) according to the manufacturer's instructions.

Techniques: Immunopeptidomics, Incubation, Confocal Laser Scanning Microscopy, Expressing, Quantitative RT-PCR

Journal: Smart Molecules

Article Title: Endocytosis‐independent cytosolic entry of messenger RNA via fluorous bilayer zippering attenuating Toll‐like receptor signaling and enables ischemic tissue salvage

doi: 10.1002/smo2.70085

Figure Lengend Snippet: Revascularization in hindlimbs by local dosage of mVEGF‐encapsulating polyplexes. (a) Therapeutic scheme. (b) Anatomy of the established hindlimb ischemia model. Ligations were made in the femoral artery at the proximal and distal sites. (c) Angiogenesis in mouse hindlimbs post ligation. (d) Visualization of blood flow by Laser Speckle Flowgraphy on Day 28 post‐dosage of mVEGF therapeutics (mVEGF: 10 μg). The magnified inset images captured by intravital confocal laser scanning microscopy (CLSM), revealing vasculature details by intravenous dosage of FITC‐dextran (MW: 10 kDa). (e) Estimation of blood perfusion volume based on quantification by laser speckle flowgraphy on Day 28 post‐dosage of mVEGF therapeutics (mVEGF: 10 μg). The data were represented as the mean ± standard deviations (s.d.) ( n = 5). (* p < 0.05, ** p < 0.01, student t test). (f) Quantification of the expressed VEGF protein on day 4 post dosage by ELISA.

Article Snippet: Culture supernatants (100 μL/well) were harvested and assayed for IFN‐α using the Mouse IFN‐α ELISA Kit (PBL Interferon Source) according to the manufacturer's instructions.

Techniques: Ligation, Confocal Laser Scanning Microscopy, Enzyme-linked Immunosorbent Assay