Additional file 1 of Inhibition of UTX/KDM6A improves recovery of spinal cord injury by attenuating BSCB permeability and macrophage infiltration through the MLCK/p-MLC pathway
Creators: name: Xie, Yong
name: Luo, Zixiang
name: Peng, Wei
name: Liu, Yudong
name: Yuan, Feifei
name: Xu, Jiaqi
name: Sun, Yi
name: Lu, Hongbin
name: Wu, Tianding
name: Jiang, Liyuan
name: Hu, Jianzhong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Mental Health

description: Additional file 1: Figure S1. a Representative fluorescence images of spinal cord specimens from EB leakage experiments at different time points before and after SCI in WT mice. Scale bar, 500 μm. b, c Quantitative evaluation of EB leakage area and fluorescence intensity in (a). Each group n=3. d Western blotting analysis of the TJs-related protein levels including ZO-1, Occludin, and Claudin-5 in the sham and SCI 3d groups of WT mice. e Quantitative analysis of the expression levels of ZO-1, Occludin, and Claudin-5 in (d). n=3 per group. f qRT-PCR verification of the mRNA levels of Claudin-5, Occludin, and ZO-1 in the sham and SCI 3d groups of WT mice. n=3 per group. Data are represented as mean ± SEM. *P&lt;0.05, **P&lt;0.01. Figure S2. The TJs structure of SCMECs was disrupted after OGD. a Representative morphological images of SCMECs. Scale bar, 20 μm. b Representative immunofluorescence images of CD31+ ECs (green) and DAPI (blue) staining. Scale bar, 10 μm. c Representative flow cytometric Histogram of SCMECs with surface marker CD31. d Schematic diagram of Transwell FITC-dextran permeation assay. upper layer, vascular endothelial cells. e, f FITC-dextran transports assay the permeability of WT SCMECs at pre- and post-OGD. n=3 per group. g Representative immunofluorescence images of TJs-related protein (Claudin-5, Occludin, and ZO-1) in the WT SCMECs when exposed to OGD. Scale bar, 20μm. h Quantitative evaluation of the fluorescence intensity of Claudin-5, Occludin and ZO-1 in (g). n=6 per group. i Quantitative evaluation of intercellular distance in (g). n=6 per group. j qRT-PCR verification of the mRNA levels of Claudin-5, Occludin, and ZO-1 in the WT SCMECs when exposed to OGD. Data are represented as mean ± SEM. ns P＞0.05, **P&lt;0.01. Figure S3. a Schematic diagram of transgenic mice breeding. b Western Blotting to identify the expression levels of UTX in UTXf/f and UTX−/− ECs. c Quantification of the expression level of UTX in (b). Each group n=3. d Representative fluorescence images of spinal cord specimens from EB leakage experiments at different time points before and after SCI in UTXf/f and UTX−/− mice. Scale bar, 500 μm. e, f Quantitative evaluation of EB leakage area and fluorescence intensity in (d). n=3 per group. g Western blotting analysis of the TJs-related protein levels including ZO-1, Occludin, and Claudin-5 in UTXf/f and UTX-/- mice at sham and SCI 3d. h Quantitative analysis of the expression levels of ZO-1, Occludin, and Claudin-5 in (g). n=3 per group. I–m qRT-PCR verification of the mRNA levels of Claudin-5, Occludin, and ZO-1 in UTXf/f and UTX−/− mice at sham and SCI 3d. n=3 per group. Data are represented as mean ± SEM. ns P＞0.05, *P&lt;0.05, **P&lt;0.01. Figure S4. a Representative fluorescent images of SCMECs after successful transfection with lentivirus. Scale bar, 100 μm. b Representative fluorescent images of different time points after intrathecal injection of lentivirus into the spinal cord. Scale bar, 500 μm. c Quantitative evaluation of GFP fluorescence intensity in (b). n=3 per group. d Representative fluorescence images of spinal cord specimens from EB leakage experiments at SCI 3d in UTXf/f, UTX-/-, UTX-/-+LV CON, and UTX−/−+LV MLCK-OE mice. Scale bar, 200 μm. e, f Quantitative evaluation of EB leakage area and fluorescence intensity in (d). n=3 per group. Data are represented as mean ± SEM. ns P＞0.05, *P&lt;0.05, **P&lt;0.01. Figure S5. a Representative phalloidin fluorescence images of SCMECs. Scale bar, 20μm. b Quantitative evaluation of mean fluorescence intensity in (a). n=6 per group. Data are represented as mean ± SEM. ns P＞0.05, **P&lt;0.01.