Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exosomal miR-181d-5p Derived from Rapamycin-Conditioned MDSC Alleviated Allograft Rejection by Targeting KLF6.

doi: 10.1002/advs.202304922

Figure Lengend Snippet: Figure 5. KLF6 was the downstream target of miR-181d-5p. A) Venn diagram displaying the predicted targets of miR-181d-5p through four different databases. B) The illustration of predicted 3′-UTR binding sites for KLF6 of miR-181d-5p. C) The binding potential of miR-181d-5p with KLF6 examined using dual-luciferase activity assay. D) The transcriptional level of KLF6 in BMDMs with different conditions determined using real-time PCR. Nor, the untreated BMDMs; LPS, the BMDMs challenged with LPS (100 ng mL−1); LPS+miR-181d-5p mimic, the BMDMs co-treated with LPS (100 ng mL−1) and miR-181d-5p mimic (100 μg mL−1); LPS+miR-181d-5p mimic NC, the BMDMs stimulated with LPS (100 ng mL−1) in the presence of miR-181d-5p mimic NC (100 μg mL−1). E) The KLF6 mRNA in BMDMs with different treatments quantified by real-time PCR, as in (cf. 4B). F) The expression and location of KLF6 in LPS-challenged BMDMs in the presence or absence of Rapa-Exo and miR-181d-5p inhibitor, as in (cf. 4B). Scale bar 50 μm. G) The mRNA levels of KLF6 in corneal allografts evaluated through real-time PCR (n = 3 per group, in pools of three samples). Allo+PBS, the allogeneic mice with the subconjunctival treatment of PBS; Allo+Rapa-Exo, the allogeneic mice topically treated with Rapa-Exo; Allo+Rapa-Exo+miR-181d-5p antagomir, the allogeneic group topically administrated with Rapa-Exo in the presence of miR-181d-5p antagomir. H) Immunoblotting analysis of KLF6 in corneal grafts with different treatments (n = 3 per group, in pools of three samples) (as in cf. 5G). *p < 0.05, **p < 0.01, ***p < 0.001, n.s, no significance.

Article Snippet: H&E Staining and Immunofluorescence Staining: After washing with cold PBS and fixing with 4% paraformaldehyde, the challenged BMDMs were permeabilized using 0.25% Triton X-100 for 10 min before incubation with rabbit anti-KLF6 antibody (14716-1-AP, Proteintech).

Techniques: Binding Assay, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exosomal miR-181d-5p Derived from Rapamycin-Conditioned MDSC Alleviated Allograft Rejection by Targeting KLF6.

doi: 10.1002/advs.202304922

Figure Lengend Snippet: Figure 6. Knockdown of KLF6 significantly blocked inflammatory response and delayed corneal immune rejection. A) The expression of pro-inflammatory cytokines in LPS (100 ng mL−1) -stimulated BMDMs in the presence or absence of siKLF6 (100 μg mL−1) for 24 h, respectively. B) The protein levels of IL-6 and TNF-𝛼in supernatants of LPS-stimulated BMDMs in the presence or absence of siKLF6 for 24 h. C) The representative slit-lamp photographs of corneal allografts treated with or without siKLF6 on postoperative day 20 (n = 3 per group). D) The survival of corneal allografts with different treatments evaluated by Kaplan–Meier approach (n = 6–8 per group). E) The pathological alterations of corneal allografts on postoperative day 20 examined using H&E staining (n = 3 per group). Scale bar 50 μm. F) The transcriptional levels of pro-inflammatory cytokines (IL-6, IL-12p35, IL-1𝛽, TNF-𝛼, and IL-17A) and CCR7 in allografts were quantified by real-time PCR (n = 3 per group, in pools of three samples). Allo+PBS, the allogeneic group with subconjunctival treatment of PBS; Allo+siKLF6, the allogeneic group topically administrated with siKLF6 (100 μg mL−1). *p < 0.05, **p < 0.01, ***p < 0.001, n.s, no significance.

Article Snippet: H&E Staining and Immunofluorescence Staining: After washing with cold PBS and fixing with 4% paraformaldehyde, the challenged BMDMs were permeabilized using 0.25% Triton X-100 for 10 min before incubation with rabbit anti-KLF6 antibody (14716-1-AP, Proteintech).

Techniques: Knockdown, Expressing, Staining, Real-time Polymerase Chain Reaction

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exosomal miR-181d-5p Derived from Rapamycin-Conditioned MDSC Alleviated Allograft Rejection by Targeting KLF6.

doi: 10.1002/advs.202304922

Figure Lengend Snippet: Figure 7. Illustration of Rapa-Exo alleviated allograft rejection directly through miR-181d-5p/KLF6 signaling. The miR-181d-5p in Rapa-Exo resolved the inflammatory response by directly targeting KLF6, through which Rapa-Exo inhibited the inflammation and ameliorated corneal allograft rejection.

Article Snippet: H&E Staining and Immunofluorescence Staining: After washing with cold PBS and fixing with 4% paraformaldehyde, the challenged BMDMs were permeabilized using 0.25% Triton X-100 for 10 min before incubation with rabbit anti-KLF6 antibody (14716-1-AP, Proteintech).

Techniques: