Additional file 1 of Impaired cerebral microvascular endothelial cells integrity due to elevated dopamine in myasthenic model

Additional file 1: Note S1. Sample preparation. Note S2. Mass spectrometry analysis. Note S3. Bioinformatics tools and databases. Figure S1. Cerebral microvascular extraction and identification in CFA and EAMG rats. The cerebral microvascular was confirmed by the tubular structure via A optical microscope and B HE staining. C the absence of the neuronal markers (Syp and Tubb3 genes) via qPCR. Statistical analysis was performed using t test, ***P < 0.001, ****P < 0.0001. Figure S2. The leakage of BBB in EAMG rats. The brain, spleen and kidney fluorescence, “ + ” and “−” indicated with or without Alexa flour 488 cadaverine injection (40 × magnification). Figure S3. The expression of DRD1-DRD5 and Scl6a3 genes in T cells (A) and B cells (B) of CFA and EAMG groups. Statistical analysis was conducted using t test, *P < 0.05, **P < 0.01, ns = no significance. Figure S4. The impact of SCH23390 (dopamine D1 receptor antagonist) and Haloperidol (dopamine D2 receptor antagonist) on bEnd.3 cells was investigated. A, B The proliferative effects of SCH23390 and Haloperidol on bEnd.3 cell line were assessed using CCK-8 assays. (C-D) The expression of CD31 was significantly increased upon incubation with both SCH23390 and Haloperidol, while Claudin5 remained unchanged. Additionally, the expression levels of Wnt3a in bEnd.3 cell line were examined through western blot, showing no significant changes in SCH23390 and Haloperidol treated cell lines. However, the results examined through western blot also showed elevated levels of p-GSK3β and active-β-catenin in SCH23390-treated cell line. Statistical analysis was conducted using one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = no significant difference. Figure S5. The effects of dopamine on T and B cells were examined. A Upon dopamine incubation, the mean fluorescence intensity (MFI) level of Th17 cells were significantly increased, whereas the MFI of Th1 and Treg cells exhibited no significant changes. B The dopamine exposure resulted in a significant increase in the MFI of CD86+ B cells and CD69+ B cells. Conversely, the MFI of CD80+ B cells decreased, and MHC II+ B cells remained unsignificant. Statistical analysis was conducted using t test, *P < 0.05, **P < 0.01, ns = no significant difference. Figure S6. Diaphragm samples were analyzed using immunofluorescence and HE staining. We performed A α-BTX and DAPI staining and B HE staining in the CFA group. We also performed C α-BTX and DAPI staining and D HE staining in the EAMG group. Blue staining represents DAPI, and α-BTX is the red staining highlighted by the white arrow. Figure S7. The effect of SCH23390 (dopamine D1 receptor antagonist) on EAMG rats was investigated by gavage every other day after the first immunization in vivo. A Clinical scores. B Survival probability and number at risk. Table S1. The criterion for each EAMG score is listed below. Table S2. The rat primer sequences are listed below. Table S3. The mouse primer sequences are listed below. Table S4. The detailed statistical results