Additional file 1 of Effects of fasudil on blood–brain barrier integrity
Creators: name: Sato, Kei
name: Nakagawa, Shinsuke
name: Morofuji, Yoichi
name: Matsunaga, Yuki
name: Fujimoto, Takashi
name: Watanabe, Daisuke
name: Izumo, Tsuyoshi
name: Niwa, Masami
name: Walter, Fruzsina R.
name: Vigh, Judit P.
name: Santa-Maria, Ana Raquel
name: Deli, Maria A.
name: Matsuo, Takayuki
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology

description: Additional file1: Table S1. Antibodies used for immunohistochemistry and western blot. Figure S1. A. The effect of fasudil (1, 10 and 100 µM, 24-h treatment) on the metabolic activity of primary confluent cultures of rat brain endothelial cells measured by MTT assay. Data are shown as mean ± SD; n=14-16 parallels/group; two separate experiments; statistical analysis: ANOVA and Dunnett test. ***p&lt;0.001 compared to the control group. B. Phase contrast micrographs from brain endothelial cells at the end of the MTT assay before cell lysis. C: control group treated with culture medium; 1, 10, 100 and 100 µM: cells treated with 1, 10 or 100 µM fasudil. Scale bar: 100 µm. Figure S2. Effect of fasudil (1 and 10 µM) treatment on the cell growth of primary rat brain endothelial cells kept in monoculture on 96-well E-plate measured by impedance kinetics. All data are presented as mean ± SD, n=13-17 parallels/groups. Statistical analysis: one-way Anova with Dunnett post-test. No statistically significant change was found between the groups. Figure S3. Effects of fasudil treatment (1 and 10 µM, 30 min) on the cellular morphology of subconfluent mono-cultures of primary rat brain endothelial cells. Representative phase contrast images at the 0 and 30 min time points. Arrows indicate the same cells in the image pairs. Scale bar: 50 µm. Figure S4. Effects of fasudil treatment (1 and 10 µM, 4 hours) on the cellular morphology of subconfluent mono-cultures of primary rat brain endothelial cells. Representative phase contrast images at the 0 and 4-hour time points. The image pairs do not show the exact same fields. Scale bar: 50 µm. Figure S5. Effects of fasudil treatment (1 and 10 µM, 24 hours) on the cellular morphology of subconfluent mono-cultures of primary rat brain endothelial cells. Representative phase contrast images at the 0 and 24-hour time points. The image pairs do not show the exact same fields. Scale bar: 50 µm. Figure S6. Effects of fasudil treatment (1 and 10 µM, 4 and 24 hours) on the cellular morphology of subconfluent mono-cultures of primary rat brain endothelial cells stained with phalloidin-A488. Representative confocal microscopy images. Green: F-actin, blue: cell nuclei, scale bar: 15 µm. White arrows: thin and continuous F-actin staining at the cell-cell junctions. Figure S7. Effects of fasudil treatment (1 and 10 µM, 4 and 24 hours) on the cellular morphology of confluent mono-cultures of primary rat brain endothelial cells stained with phalloidin-A488. Representative confocal microscopy images. Green: F-actin, blue: cell nuclei, scale bar: 15 µm. White arrows: thin and continuous F-actin staining at the cell-cell junctions. Figure S8. a. Effect of fasudil (1 µM) on cell viability without and with U-46619 (20µM) treatment. Cell viability was measured by the cell counting kit-8 WST-based assay test on pimary rat brain endothelial cells kept in monoculture BBB model. Data is presented as mean ± SEM. b. Effects of fasudil (1 µM) on expression of claudin-5 in a triple co-culture model with primary brain pericytes and astrocytes without and with U-46619 (20 µM) treatment. Arrows indicate the changes in claudin-5 distribution. Bar: 50 µm.