Additional file 1 of si-Tgfbr1-loading liposomes inhibit shoulder capsule fibrosis via mimicking the protective function of exosomes from patients with adhesive capsulitis
Creators: name: Sun, Yaying
name: Luo, Zhiwen
name: Chen, Yisheng
name: Lin, Jinrong
name: Zhang, Yuhan
name: Qi, Beijie
name: Chen, Jiwu
Publisher: name: figshare
Year: 2022
Subjects: Biophysics
Biochemistry
Cell Biology
Molecular Biology
Pharmacology
Chemical Sciences not elsewhere classified
Developmental Biology
Cancer
Mental Health

description: Additional file 1: Fig. S1. Flowchart of animal experiments. One week after model establishment, two intra-articular injections were delivered weekly. Samples were harvested at 21d from model establishment. Fig. S2. Fibrotic changes was observed in human capsule samples in group S. Representative immunofluorescence images of α-SMA (A) and Col 1 (B) in the human capsular samples. Bar=100 μm. Fig. S3. Exo-NS and Exo-S were uptaken by CDFs. Typical image of the uptake of PKH67-labeled Exo-NS and Exo-S (green) by CDFs (Dapi blue) and negative control (Dye-only). Bar=25 μm. Fig. S4. TGF-β highly expressed in human capsule samples in group S than group NS. Typical immunofluorescent images illustrated the expression of TGF-β (green) in the human shoulder capsular samples. Scale bar=100 μm. Fig. S5. Exo-NS had no significant influence on TGF-β mediated promotion of cell viability. Viability of CDFs by CCK-8 analysis. ***: P &lt; 0.001 compared to group NC. Fig. S6. Exo-S were uptaken by NIH3T3 cells. Typical picture of the uptake of PKH67-labeled Exo-S (green) by NIH3T3 (Dapi blue) and negative control (Dye-only). Bar=25 μm. Fig. S7. Exo-S relieved fibrogenesis of NIH3T3 cells induced by TGF-β. A and B: Protein level of Col 1 and α-SMA in NIH3T3 cells under different stimulations and quantification; C and E: Proliferation of NIH3T3 cells and quantification. Bar=180 μm; D and F: Migration ability of cells (red dotted line indicated the border of wound) and quantification; G: Viability of NIH3T3 cells probed by CCK-8 assay. ***: P &lt; 0.001 compared to group NC; ****: P &lt; 0.0001 compared to group NC; #: P&lt;0.05 compared to group TGF-β; ##: P&lt;0.01 compared to group TGF-β; ###: P&lt;0.001 compared to group TGF-β; ####: P&lt;0.0001 compared to group TGF-β. Fig. S8. miR-142 inhibited CDFs fibrogenesis induced by TGF-β. A and C: Proliferation of CDFs and quantification. Bar=180 μm; B and D: Migration ability of CDFs (orange dotted line indicated the border of the wound) and quantification; E: Viability of CDFs determined by CCK-8 assay; F and G: Protein level of p-Smad2/3 and t-Smad2/3 in CDFs and quantification; H and I: Collagen contraction ability of CDFs and quantification (red dotted circle indicated the collagen). **: P &lt; 0.01 compared to group NC; ***: P &lt; 0.001 compared to group NC, ****: P &lt; 0.0001 compared to group NC; ##: P &lt; 0.01 compared to group TGF-β + NC mimics; $: P &lt; 0.05 compared to group NC inhibitor; $$: P &lt; 0.01 compared to group NC inhibitor. Fig. S9. Anti-fibrotic effect of Exo-S depended on miR-142 in vitro. A and B: Proliferation of CDFs and quantification. Bar=180 μm; C: Viability of CDFs was determined using CCK-8 assay; D and E: Immunofluorescent staining of α-SMA in CDFs (red forα-SMA and blue for nucleus) and quantification. Bar=25μm. *: P &lt; 0.05 compared to group TGF-β, **: P &lt; 0.01 compared to group TGF-β; #: P &lt; 0.05 compared to group TGF-β + Exo-S. Fig. S10. Expression of miR-142 and Tgfbr1 in patients shoulder capsules. The expression of miR-142 (A) and Tgfbr1 (B) in patients’ capsule samples were lower in group S than in group NS. **: P &lt; 0.01; ****: P &lt; 0.0001. Fig. S11. The efficacy of siRNAs for knocking down the expression of Tgfbr1 in NIH3T3 cells. A and B: The knock-down efficacy of three siRNAs and quantification; C and D: Proliferation of NIH3T3 cells and quantification. Bar=180 μm; E: Relative expression of fibrotic markers in NIH3T3 cells; F and G: Migration ability of NIH3T3 cells (red dotted line indicated the border of wound) and quantification. *: P &lt; 0.05 compared to group NC; **: P &lt; 0.01 compared to group NC; ***: P &lt; 0.001 compared to group NC; ****: P &lt; 0.0001; #: P &lt; 0.05 compared to group TGF-β; ##: P &lt; 0.01 compared to group TGF-β; ###: P &lt; 0.001 compared to group TGF-β. Fig. S12. Tracing of DiO-labeled liposomes in vitro. Liposomes entered NIH3T3 cells when co-culturing for 30min and 60min. Fig. S13. Tracing of liposomes in vivo. A: 24h following intra-articular injection, DiO-labeled liposomes were viewed in the cells of capsule tissue. Bar=25μm; B: Immediately, one day, and two days after DiR-labeled liposomes injection, fluorescent signaling was viewed at the injection site (white circle). Fig. S14. Toxicity evaluation in vivo. Typical HE picture of the major organs (liver, lung, intestine, heart, spleen, and kidney) in mice model with or without si-Tgfbr1-loading liposomes injections. Table S1. Primary antibodies used in the experiments. Table S2. The sequence of miRNAs and siRNAs. Table S3. Primers used in the study. Table S4. Basic characteristics of patients enrolled.