Fig 2: PCR array analysis and validation of genes differentially expressed between shNT and shCSNK1D cells(A) Scatter plot of gene expression analysis by RT2 Profiler PCR Array in the shNT MDA-MB-231 cells compared to shCSNK1D cells. The central line indicates unchanged gene expression. (B) Taqman validation of genes identified in the RT Profiler array. The histogram represents fold change of genes up-or down-regulated in shCSNK1D compared to shNT control cells. (C) Western blot analysis of the expression of occludin (OCLN), junction adhesion molecule A (F11R) and claudin-1 (CLDN-1) in control (shNT) and inactivated cell lines (shCSNK1D). (D) Detection of OCLN and F11R by immunofluorescence in shCSNK1D compared to control shNT cells. Nuclei were stained with Dapi. Arrows indicate the increased expression of tight junction proteins.

Fig 3: Ex vivo fluorescent imaging of mouse tumor xenografts.(A) Imaging of tumor xenografts at 48-h post-injection of JAM-A mAb/IR700 and controls. Each xenograft was cut into 2 halves to image internal sides of the tumors. The fluorescence was significantly higher in tumors of animals which were treated with JAM-A mAb/IR700 (Probe). Controls: 1) mix of JAM-A mAb and JAM-A mAb/R700 conjugates (2-to-1 ratio, respectively) – Probe competition; 2) non-specific mouse IgG/IR700 conjugates – Norm mAb, and 3) IR700 alone. Pseudo-color images of IR700 fluorescence are presented in the same scale. (B) Quantitative analysis of IR700 fluorescent intensities in tumor xenografts. Values represent mean of IR700 fluorescence per mm2 of internal surfaces of the tumor xenografts. The fluorescence of PC3pip and MDA-MB-231 xenografts was analyzed separately. Vertical bars – SD; n = 5 per each group, † – p<0.001 to all controls; ** – p<0.02 to Norm Ab and IR700 alone; * – p<0.05 to IR700 alone (Mann–Whitney-U test).

Fig 4: In inflammatory cortical lesions, CD4+ T cells are arrested within the PVS in the absence of astrocytic JAM-A. Asymptomatic inflammatory cortical lesions were induced in JAM-A CKO and WT mice via the microinjection of AdIL-1 into the frontal cortex. Brains were harvested for histopathology at 7 days post-injection. (A and B) Lesions in JAM-A CKO mice, as measured by the area of neuronal cell death (loss of NeuN, red, white arrows), showed a trend in smaller lesion size compared with WT mice that did not reach statistical significance (P = 0.18, CKO n = 16 mice, WT n = 10 mice, Mann–Whitney test). (C and D) CD4+ cells (green and circled in white) showed an increased but statistically non-significant trend in number within JAM-A CKO lesions, scale bar: 125 µm [average number/mm2, 14.5 (CKO) versus 11.1 (WT), P = 0.11; CKO n = 4 mice, WT n = 5 mice, Mann–Whitney test]. (E–I) In JAM-A CKO mice, a higher proportion and total number of CD4+ cells (green) co-localize to the laminin-positive (red) basement membrane of the PVS while a lower proportion of cells localize to the parenchyma (laminin-negative) though total number of cells within the parenchyma per area remain the same. (E) Scale bar: 25 µm. (F) Average proportion of CD4+ cells in the PVS over total CD4+ cells: 0.50 (CKO) versus 0.29 (WT), P = 0.02. (G) Average number CD4+ cells in the PVS/mm2: 7.6 (CKO) versus 3.4 (WT), P = 0.03. (H) Average proportion of CD4+ cells in the parenchyma over total CD4+ cells: 0.50 (CKO) versus 0.70 (WT), P = 0.02. (I) Average number CD4+ cells in the PVS/mm2: 7.2 (CKO) versus 7.6 (WT), P = 0.85. (F–I) N = 5 mice per group, Mann–Whitney tests.

Fig 5: Gene expression responses of the JAMs to varying LPS doses relative to the geometric mean of YWHAZ and ACT-ß expression (A–C) and to 10 ng/mL LPS over a 24 h time course with sampling at 0, 0.5, 1, 2 and 4 h and then at 24 h in THP-1 monocytes (D–F). (A,D) JAM-A gene expression, (B,E) JAM-B gene expression and (C,F) JAM-C gene expression. The comparisons in (A) to (C) are between the control without LPS and the different LPS doses while those in (D) to (C) are between expression at time 0 and at various times. *** indicates p = 0.0001, ** indicates p = 0.001, * indicates p = 0.05.