Additional file 1 of Endothelial cell-derived exosomes boost and maintain repair-related phenotypes of Schwann cells via miR199-5p to promote nerve regeneration
Creators: name: Huang, Jinsheng
name: Zhang, Geyi
name: Li, Senrui
name: Li, Jiangnan
name: Wang, Wengang
name: Xue, Jiajia
name: Wang, Yuanyi
name: Fang, Mengyuan
name: Zhou, Nan
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Science Policy
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Figure S1. The effect of EC-EXO on cell phenotypes of SCs. A The proliferation of SCs treated with EC-EXO of different concentration (1, 10, 50 and 100 μg/mL) for 24 and 48 h was detected by CCK8 assay. The data are expressed as mean ± SD (n = 3). B Cell growth curve was detected by CCK-8 assay in 50 μg/mL EXO-treated and control groups. The data are expressed as mean ± SD (n = 3). C SC apoptotic levels determined by flow cytometry assay. The data are expressed as mean ± SD (n = 3). D Statistical results of cell apoptotic. The data are expressed as mean ± SD (n = 3). E The protein levels of proliferation-related protein (PCNA) and apoptosis-related protein (BAX and Bcl2) were analyzed by western blot following incubation of SCs to EC-EXO for 24 h. F Quantification of PCNA, BAX and Bcl2 levels in EXO-treated and control groups. The data are expressed as mean ± SD (n = 3). G The mRNA levels of immune factors (LIF, Gal-3 and MCP-1) were detected by RT–qPCR of SCs in each group. The mRNA levels are expressed as fold change of the control. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S2. Comparison of the effects of EC-EXO and SC-EXO on the proliferation and migration phenotypes of SCs. A Representative transmission electron microscopy (TEM) images of SC-EXO. Scale bar, 100 nm. B Protein immunoblots of exosomes, including the typical markers (TSG101, CD9 and CD81) and GAPDH. C Particle size distribution of SC-EXO measured by nanoparticle tracking analysis (NTA), inset showing representative exosome images captured from the NTA video frames. D Representative EdU staining images in different groups. Scale bar, 50 μm. E Statistical evaluation of percentage of EdU-positive SCs. The data are expressed as mean ± SD (n = 3). F Representative images of vertical migration of SCs in different groups for 24 h. Scale bar, 100 μm. G The number of migrated SCs was counted and analyzed. The data are expressed as mean ± SD (n = 3). H Representative images of the colony formation in indicated groups. Scale bar, 500 μm. I Statistical results of the colony formation in each group. The data are expressed as mean ± SD (n = 3). *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S3. PI3K inhibitor (PI3Ki) could depress the proliferative and migratory phenotype of SCs induced by EC-EXO. A Western blot of PI3K, p-PI3K, AKT, p-AKT and PTEN in control, EXO and EXO + PI3Ki groups. B Quantification of PI3K, p-PI3K, AKT, p-AKT and PTEN protein levels in each group. Data are expressed as mean ± SD (n = 3). C Representative EdU staining images in different groups. Scale bar, 50 μm. D Statistical evaluation of percentage of EdU-positive SCs. The data are expressed as mean ± SD (n = 3). E Representative images of vertical migration of SCs in different groups for 24 h. Scale bar, 100 μm. F The number of migrated SCs was counted and analyzed. The data are expressed as mean ± SD (n = 3). G Representative images of the colony formation in indicated groups. Scale bar, 500 μm. H Statistical results of the colony formation in each group. The data are expressed as mean ± SD (n = 3). ns = not significant, *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S4. EC-EXO improved the anti-apoptotic ability of injured nerve tissue. To study the effect of EC-EXO in vivo, 50 μg/mL EC-EXO (20 μL) were injected locally under the epineurium of injured sciatic nerve. A The left picture is a schematic illustration of a sciatic nerve crush model on rats. The right picture illustrated the injured sciatic nerve during the surgery. B, C TUNEL-positive (green) cells were detected in sciatic nerves by TUNEL staining and the statistical results. The data are expressed as mean ± SD (n = 3). Scale bar, 20 μm. D The protein levels of BAX and Bcl2 of sciatic nerves in each group were analyzed by western blot. E Quantification of BAX and Bcl2 levels in each group. The data are expressed as mean ± SD (n = 3). *p &lt; 0.05, **p &lt; 0.01.