Additional file 1 of ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood–brain barrier integrity after traumatic brain injury
Creators: name: Meng, Shan
name: Cao, Hui
name: Huang, Yichen
name: Shi, Ziyu
name: Li, Jiaying
name: Wang, Yana
name: Zhang, Yue
name: Chen, Shuning
name: Shi, Hong
name: Gao, Yanqin
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Table S1. Primer sequences used in real-time PCR analysis. Fig. S1. Partial DNA sequencing after ASK1-K716R and p-ASK1 expressed in brain cells. A A schematic representation of the nucleotide substitution (-AAGGAAATC- to -AGAGAAATA-) at site 716, resulting in the replacement of lysine with arginine during translation. B Representative immunofluorescence images displaying the colocalization of microglia (Iba1+), astrocytes (GFAP+), and neurons (NeuN+) with p-ASK1 in cortex of both WT TBI and ASK1-K716R TBI mice. Scale bar, 50 μm. Fig. S2. Effect of ASK1-K716R on neuronal apoptosis at day 3 post-TBI. A A schematic diagram showing the regions of interest around the lesioned site. Scale bar, 50 μm. The rectangle illustrates where images were taken. B Representative images of TUNEL staining in the cortical region around the lesioned site. C Quantification of the number of TUNEL+ neurons per hemisphere (n = 5 WT sham, n = 6 ASK1-K716R sham, n = 6 WT TBI, n = 6 ASK1-K716R TBI). All data from male mice are presented as the mean ± SD. One-way ANOVA test and Bonferroni post hoc. ** p &lt; 0.01, *** p &lt; 0.001, as indicated. Fig. S3. ASK1-K716R protects tight junction proteins in endothelial cells 3 days following TBI. A Representative images of CD31+ (green) and Occludin+ (red) immunofluorescent staining. B Representative images of CD31+ (green) and VE-Cadherin+ (red) immunofluorescent staining. Scale bar, 20 μm. Fig. S4. ASK1-K716R does not alter the population of adaptive immune cell population in peripheral immune cells 3 days following TBI. A Gating strategy for immune cells in the peripheral blood and spleen. B Quantification of the numbers of immune cells in the blood. C Quantification of the numbers of immune cells in the spleen. n = 5 WT for sham, n = 6 for ASK1-K716R sham, n = 8 for WT TBI, n = 9 for ASK1-K716R TBI. All data from male mice are presented as the mean ± SD. one-way ANOVA test and Bonferroni post hoc. ns, no significance. Fig. S5. Effect of ASK1-K716R on early white matter injury at day 3 post-TBI. A A schematic diagram of the regions of interest in CTX, EC, and STR. B-C Representative images of MBP (green)/SMI32 (red) (B), and NF200 (purple) immunofluorescent staining (C). Scale bar, 100 μm. D-F Quantification of the fluorescence intensity of MBP (D), SMI32/MBP (E), and NF200 (F) in the CTX, EC, and STR. n = 4 for WT sham, n = 4 for ASK1-K716R sham, n = 7 for WT TBI, n = 7 for ASK1-K716R TBI). All data from male mice are presented as the mean ± SD. one-way ANOVA test and Bonferroni post hoc. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001, ns, no significance, as indicated. Fig. S6. Spatial learning and memory assessment 30 to 34 days after TBI using the Morris water maze. A Representative traces in the learning phase (upper panel) and memory test (lower panel). B Escape latency to find the hidden platform in the learning phase. n = 8 for WT sham, n = 8 for ASK1-K716R sham, n = 12 for WT TBI, n = 12 for ASK1-K716R TBI. All data from male mice are presented as the mean ± SD. Two-way ANOVA repeated measurement. * p