MOESM1 of Meningeal inflammation changes the balance of TNF signalling in cortical grey matter in multiple sclerosis

Additional file 1: Figure S1. Tissue blocks were taken from the precentral gyrus of 1 cm thick coronal slices of post-mortem MS brains that had been dissected into 2cmx2cm blocks and snap frozen. Sections from the bocks were then subjected to anti-MOG immunohistochemistry to identify areas of subpial demyelination and NAGM. The areas of tissue were then dissected out and processed as shown in the flow diagram. Figure S2. A. Supervised hierarchical clustering of 4658 transcripts showing a significant alteration in any of the groups vs control samples from healthy subjects (p<0.01 Fold Change>1.5 vs Control group). A false discovery rate (FDR) assessment was performed by permutating MS and control samples according to the table shown in B. 5 MS and 5 control samples were included in each of two groups and FDR analysis was conducted by comparing the statistically different genes between the two groups. Different stringency conditions were tested. The table in panel C. shows for each tested condition (p<0.05-0.01; fold change 1.5-2), the resulting numbers of real differentially expressed transcripts (Experimental), the median number of probe sets derived from the permutation analysis (FRD#) and the percentage of FDR with respect to the real values (FDR%). A plot of the results showing p-value/fold cutoff on the x axis, FDR% on the left axis and Experimental values on the right axis is also shown (D). Figure S3. A: List of several genes of neuronal/axonal components downregulated in the GML of SPMS respect to healthy controls as revealed by microarray gene expression analysis. B-G: Real-time RT-PCR using the same RNA samples employed in the microarray analysis was performed in order to validate the greatest deregulated gene expression in the motor cortex of MS group compared to controls including: decreased expression of neuronal genes, as the neuronal genes for parvalbumin (PVALB, B) and the large (C), medium (D) and low (E) molecular weight neurofilament proteins (NEFL, NEFM NEFH); increased expression of activated microglia markers, CD68 (F) and inducible NO synthase (iNOS, G) was measured by Real-time RT-PCR. For each statistical comparison the p-value, obtained by non-parametric Mann-Whitney test, has been reported (***p<0.001, **p<0.01, *p<0.05).