Journal: Theranostics

Article Title: Exosome-mediated delivery of RBP-J decoy oligodeoxynucleotides ameliorates hepatic fibrosis in mice.

doi: 10.7150/thno.69885

Figure Lengend Snippet: Figure 1. RBP-J decoy oligodeoxynucleotides (ODNs) inhibited Notch signaling activation. (A) The structure of the decoy ODNs for the transcription factor RBP-J. (B–D) RAW264.7 cells or HUVECs were transfected with RBP-J decoy ODNs (Decoy RBP-J) or control decoy ODNs (Decoy Ctrl). After 24 h, Notch signaling was activated by the overexpression of the Notch intracellular domain (NICD) (Ad-NICD) in RAW264.7 cells or with Dll4-Fc in HUVEC cells. The mRNA levels of Notch target genes were detected by qRT-PCR in RAW264.7 cells (B) or HUVECs (C). The expression of Hes1 in RAW264.7 cells was determined by western blot, with β-actin serving as a reference control (D). (E) Luciferase reporter assay. HeLa cells were co-transfected with the RBP-J reporter plasmid pGa9816, pEFBOS-NICD, and increasing concentrations of RBP-J decoy ODNs; pRL-TK served as a reference control. Luciferase activity was detected 24 h after transfection. (F) Representative electrophoretic mobility shift assay (EMSA) results showing the binding of RBP-J to RBP-J decoy ODNs. HeLa cells were transfected with pCMV-RBP-J-Flag. Next, cell extracts were incubated in EMSA/Gel Shift binding buffer in the presence of biotin-labeled RBP-J decoy ODNs, unlabeled RBP-J decoy ODNs, control decoy ODNs, or an anti-Flag antibody, as indicated. Bars = means ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The samples were analyzed by sodium dodecyl sulfate (SDS)–PAGE, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and incubated first with primary antibodies targeting Hes1, CD9, Alix, flotillin-1 (Cell Signaling Technology), HEY1, β-actin (Proteintech, Wuhan, China), and VDAC1 (Abcam, Cambridge, UK) and then with HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Zhuangzhi Bio, Xi’an, China).

Techniques: Activation Assay, Transfection, Control, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Reporter Assay, Plasmid Preparation, Activity Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Gel Shift, Labeling

Journal: Theranostics

Article Title: Exosome-mediated delivery of RBP-J decoy oligodeoxynucleotides ameliorates hepatic fibrosis in mice.

doi: 10.7150/thno.69885

Figure Lengend Snippet: Figure 4. Exosomes loaded with RBP-J decoy oligodeoxynucleotides (ODNs) inhibited Notch signaling in macrophages. Bone marrow-derived macrophages (BMDMs) were incubated with Dil-exosomes (A) or exosomes loaded with FAM-labeled ODNs (B) for 24 h and then analyzed by fluorescence microscopy. Nuclei were counterstained with Hoechst 33258. (C) Exosomes were loaded with oligonucleotides (miR-140-5p mimics or control mimics, 100 pmol) by electroporation and different concentrations of the loaded exosomes were incubated with BMDMs. After 24 h, the level of miR-140-5p was quantified in the BMDMs by qRT-PCR, with U6 serving as a reference control. (D–F) BMDMs were incubated with exosomes that had been electroporated with RBP-J decoy or control decoy ODNs for 24 h and then stimulated with LPS (200 ng/mL) for 24 h. The mRNA levels of Hes1, IL1β, IL6, and iNOS in BMDMs were detected by qRT-PCR (D). The protein level of Hes1 was determined by western blot (E), with β-actin serving as a reference control. The levels of IL1β and IL6 in culture supernatant were detected by ELISA (F). (G, H) Mice were intraperitoneally injected with CCl4 twice a week for 6 weeks; exosomes loaded with RBP-J decoy or control decoy ODNs (exosomes/decoy ODNs = 200 ng/2.5 nmol) were infused into mice four times via tail vein injection. F4/80+ hepatic macrophages were isolated using magnetic-activated cell sorting (MACS). The protein level of Hes1 was determined by western blot (G) and the mRNA levels of Hes1, Cyld, IL1β, and IL6 in F4/80+ hepatic macrophages were measured by qRT-PCR (H). Bars = means ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The samples were analyzed by sodium dodecyl sulfate (SDS)–PAGE, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and incubated first with primary antibodies targeting Hes1, CD9, Alix, flotillin-1 (Cell Signaling Technology), HEY1, β-actin (Proteintech, Wuhan, China), and VDAC1 (Abcam, Cambridge, UK) and then with HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Zhuangzhi Bio, Xi’an, China).

Techniques: Derivative Assay, Incubation, Labeling, Fluorescence, Microscopy, Control, Electroporation, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Isolation, FACS

Journal: Journal of nanobiotechnology

Article Title: ROS-responsive liposomes as an inhaled drug delivery nanoplatform for idiopathic pulmonary fibrosis treatment via Nrf2 signaling.

doi: 10.1186/s12951-022-01435-4

Figure Lengend Snippet: Fig. 7 DTP@DMF NPs suppress fibrosis and macrophage accumulation in lung tissue via Nrf2 signaling. a Representative H&E and Masson’s trichrome staining and b the severity score of fibrosis after DMF, DP@DMF NPs and DTP@DMF NPs treatment (n = 5). c α-SMA and d collagen Ia1 protein levels in fibrotic tissue (n = 3). e Nrf2 and f HO-1 levels in fibrotic tissue (n = 3). g The Nrf2 (red) and HO-1 (red) expression in F4/80+ macrophages (green) detected by immunofluorescence analysis. h Immunohistochemistry of F4/80+, CD86+, and CD206+ macrophages in pulmonary tissues, and i–m quantification of total macrophages and M1 and M2 phenotypes (n = 5). n TGF-β levels in BALF, o SOD and p MDA levels in tissue (n = 5). Statistical analyses were performed via one-way ANOVA with S–N-K post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Antibodies against Nrf2, collagen Ia1, α-smooth muscle actin (SMA), F4/80, and CD206 were purchased from Cell Signaling Technology (MA, USA).

Techniques: Staining, Expressing, Immunofluorescence, Immunohistochemistry