Additional file 1 of Global alterations to the choroid plexus blood-CSF barrier in amyotrophic lateral sclerosis

Additional file 1: Figure S1. ZO-1 immunofluorescence was performed on control (n = 4) and ALS CP (n = 5) and staining was imaged under 20x using confocal microscopy. DAPI denotes nuclei and DIC (differential interference contrast) images were taken to show tissue morphology. Scale bar: 50um. Red arrowheads point to areas of loss of ZO-1 staining in between CP epithelial cells. Figure S2. A) Immunohistochemistry for Claudin 5 was performed on control (n = 3) and ALS CP (n = 5). Pictures were taken at 40X magnification. Scale bar: 20 μm. Black Arrows point to staining in endothelial cells and red arrowheads point to staining within the lumen of blood vessels. B) Western blot analysis was performed on 9 controls and 18 SALS CP lysates and membranes were probed for Claudin 3 and 5, as well as Occludin. GAPDH was used as a loading control. Figure S3. A) Quantification of immunofluorescence signal intensity for CD13 and PDGFRbeta on n = 3 control and n = 5 ALS CP samples was performed using Imaris. Image stacks were taken at 63x magnification and ten different Z-stack fields per sample were captured and quantified. Tissues boundaries were delineated and signal intensity was normalized to section volume. The asterisk denotes significance with a p-value of 0.0095. Values for PDGFRbeta were not found to be significant. B) CD13/ANPEP immunohistochemistry in ALS (n = 5) and control (n = 4) CP. Black arrows point to CD13 stain around blood vessels on controls and red arrowheads denote vascular areas of CD13 loss in ALS. Figure S4 A) CD3 staining in ALS-CP tissues. Scale bar: 20 μm. B) MERTK immunohistochemistry in ALS and Control CP. Pictures were taken at low magnification to show extent of MERTK expression. Scale bar 100 μm C) p-TDP43 (S409/410) staining in CP from ALS and Control postmortem tissues. Only two ALS cases showed any inclusions, and these were located in the choroid stroma. Scale bar: 20 μm. Figure S5 Heatmap showing Z-scores across all samples for all gene targets that had reliable MSD signal. Samples are presented in their respective groups (control vs ALS), ranked by cumulative sum of all analytes measured. Figure S6 All cytokines and chemokines analyzed by MSD immunoassays in n = 22 controls and n = 44 ALS-CP lysates. None of the shown analytes showed significant differences between the two groups through a logistic regression analysis that accounts for patient age (under 65 vs above 65), as well as the plate on which the sample was run to avoid batch effect bias. Both IFN-γ and TNF-α had low signal to background ratio. Supplemental Table 1. ToppGene Analysis showing the top 30 enriched Biological Processes. Supplemental Table 2. MSD immunoassay analysis of CP lysates from ALS and non-neurological disease controls. Supplemental Table 3. Demographics for post-mortem tissue samples. Supplemental Table 4. CP samples used for RNA-seq. Supplemental Table 5. List of antibodies used in analysis of CP samples.