Additional file 1 of Agomir-122-loaded nanoparticles coated with cell membrane of activated fibroblasts to treat frozen shoulder based on homologous targeting
Creators: name: Peng, Zhen
name: Qi, Beijie
name: Luo, Zhiwen
name: Sun, Yaying
name: Zhang, Xingyu
name: Lin, Jinrong
name: Pang, Jinhui
name: Zhang, Peng
name: Zhao, Zhihu
name: Wang, Xianwen
name: Chen, Jiwu
Publisher: name: figshare
Year: 2024
Subjects: Biochemistry
Space Science
Medicine
Cell Biology
Neuroscience
Pharmacology
Biotechnology
Chemical Sciences not elsewhere classified
Biological Sciences not elsewhere classified

description: Additional file 1: Figure S1. The level of p-Smad2/3 relative to t-Smad2/3 in CDFs with different interventions. (n = 3). **: p &lt; 0.01; ****p &lt; 0.0001. Figure S2. Agomir-122@MNP inhibited TGF-β-induced activation of CDFs. A Cell proliferation of CDFs with different interventions, as determined by BrdU staining (green). B Quantification of BrdU staining. C Cell activity of CDFs with different interventions, as determined by CCK-8 assay. D Cell contraction ability of CDFs with different interventions, as determined by collagen gel contraction assay. E Quantification of collagen gel area. Scale bar: 200 μm. (n = 3). *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; ****p &lt; 0.0001. Figure S3. Immunofluorescence of Col 1 (green) and α-SMA (red) expression on NIH3T3 cells (nuclei in DAPI blue) challenged with TGF-β (A), and quantification (B) (n = 3). *p &lt; 0.05; **p &lt; 0.01. Figure S4. Characterization of Agomir-122 loaded nanoparticles wrapped with cell membrane of activated NIH3T3 cells. A The level of α-SMA in NIH3T3 cells with TGF-β stimulation, determined by western blot, and quantification (n = 3). B SDS-PAGE analysis of NIH3T3 cells, Agomir-122MmNP, and Agomir-122@NP. C Morphology of Agmoir-122@NP and Agomir-122@MmNP, observed by STM. Scale bar: 100 nm. D Diameters of Agomir-122@NP and Agomir-122@MmNP (n = 3). E Zeta potentials of Agomir-122@NP and Agomir-122@MmNP (n = 3). **p &lt; 0.01. I: NIH3T3, II: Agomir-122MmNP, III: Agomir-122@NP. Figure S5. Determining a concentration of Agomir-122 effective in up-regulating miR-122 in NIH3T3 cells (A), and the effectiveness of Agomir-122@NP and Agomir-122@MmNP in up-regulating miR-122 level, as compared to Agomir-122 alone, in NIH3T3 cells (B). (n = 3). **p &lt; 0.01; ***p &lt; 0.001. Figure S6. The biosafety of different agents on NIH3T3 cells, as measured by apoptosis flow cytometry. (n = 3). Figure S7. The uptake of Agomir-122@NP or Agomir-122@MmNP (DiR-labeled) by NIH3T3 cells stimulated or not stimulated with TGF-β, and quantification. (n = 3). Scale bar: 25 μm. ***p &lt; 0.001. NS no significance. Figure S8. Immune evasion of Agomir-122@MmNP. Raw264.7 cells with internalized Agomir-122@NP or Agomir-122@MmNP were detected by flow cytometry, with quantification. (n = 3). ***p &lt; 0.001; ****p &lt; 0.0001. Figure S9. Agomir-122@MmNP inhibited TGF-β-induced activation of NIH3T3 cells. A Cell proliferation of NIH3T3 cells with different interventions, as determined by BrdU staining (green). B Quantification of BrdU staining. C Cell activity of NIH3T3 cells with different interventions, as determined by CCK-8 assay. D Cell contraction ability of NIH3T3 cells with different interventions, as determined by collagen gel contraction assay (red circle indicated the collagen gel). E Quantification of collagen gel area. Scale bar: 200 μm. (n = 3). I: NC, II: TGF-β, III: TGF-β + Agomir-122@NP, IV: TGF-β + Agomir-122@MmNP. *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; ****p &lt; 0.0001. Figure S10. Evaluate the impact of Agomir-NC, NP, and MNP on activated NIH3T3 cells. A Assess intergroup cell proliferation capability through BrdU(green) staining. B Compare the vitality of NIH3T3 cells among different groups using CCK-8. C Statistical chart of BrdU-positive cells from BrdU staining. (n=3). Scale bar: 200 μm. ns no significance, NC Negative control. Figure S11. Representative live tracing image of agent injection into the index shoulder. One day, 3 days, 5 days, and 7 days after DiR-labeled Agomir-122@MmNP injection, fluorescent signaling was viewed at the injection site in a diminishing manner. Figure S12. Therapeutic effect of Agomir-122@MmNP against frozen shoulder in a mouse model. A Schematic chart of model establishment, intervention, as well as sample harvest and observation. B Representative histological image of mouse shoulder joint capsule with different interventions, as illustrated by HE and α-SMA staining (α-SMA in red and DAPI in blue). C Quantification of cell counts in joint capsule of different groups. D Quantification of joint capsule thickness of different groups. E α-SMA expression in joint capsule of different groups. F Passive ROM of the index shoulder of different groups. Scale bar: 200 or 100 μm. (n = 6). *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; ****p &lt; 0.0001. Figure S13. HE staining of the main organs (heart, liver, spleen, lung, kidney and Intestine) after treatments. Scale bar: 100 μm.