Fig 2: Old World (OW) mammarenaviruses do not alter integrity of model intestinal epithelia during infection. (A) Diagram of Caco-2 cell seeding for apical infection (left) or basolateral infection (right) during polarization; (B) After 21-day polarization period, Caco-2 cells form confluent monolayers with markers of polarization such as apical tight junction protein ZO-3 (green), and a-DG (red) on the basolateral side of cells; (C) Caco-2 cells were seeded for apical infection (top) or basolateral infection (below) and polarized for 21 days. After which the cells were either Mock infected with PBS, or lymphocytic choriomeningitis virus (LCMV)-Armstrong, LCMV-WE, ML-29, Mopeia (MOPV), or Venezuelan Equine Encephalitis (VEE) virus (vaccine strain TC-83) at an MOI of 0.3 PFU/cell. TEER measurements were taken daily for 5 days. Values shown are the means of 3 replicates, with the error bar representing the standard deviation of the means.

Fig 3: Effect of Tβ4 on LPS-induced relative mRNA expression of tight junction genes in human brain microvascular endothelial cells. Cells were exposed to LPS at 100 ng/ml for a period of 24 h. The mRNA expression of (A) ZO1, (B) ZO3 (C) Occludin and (D) Claudin 5 were measured using reverse transcription-quantitative PCR. (E) Representative image of the western blotting analysis of the Occludin protein expression level. (F) Semi-quantification of the occludin western blot. The experiments were replicated 3-5 times and amplifications were performed and normalized to GAPDH. Results are presented as the mean ± SEM. *P<0.05 vs. untreated cells (control). #P<0.05 vs. LPS treated cells. NS, non-significant; ZO, zonula occludens; LPS, lipopolysaccharide; Tβ4, thymosin β4.

Fig 4: Effect of Nrg1 antibody on LPS-stimulated ZO3 and occludin in hBMVECs. Cultured hBMVECs were pre-treated with Nrg1 antibody for 2 h, Tβ4 for 2 h and stimulated with LPS for 24 h. Dual-staining immunofluorescence analysis of (A) ZO3 and (B) occluding (green staining; DAPI, blue staining). (C) Semi-quantification of number of green fluorescence-positive cells. Objective magnification, x20. hBMVECs, human brain microvascular endothelial cells. ZO, zonula occludens; Nrg1, neuregulin-1; hBMVECs, human brain microvascular endothelial cells; LPS, lipopolysaccharide. Results are presented as the mean ± SEM. *P<0.05 vs. untreated cells (control). #P<0.05 vs. LPS treated cells.

Fig 5: Effect of Tβ4 on ZO1, ZO3, occludin and p65 expression in hBMVECs stimulated with LPS. Cultured hBMVECs were pre-treated with Tβ4 for 2 h and then stimulated with LPS for 24 h. Dual staining immunofluorescence analysis of (A) ZO1, (B) ZO3 and (C) Occludin (green staining; DAPI, blue staining). (D) Semi-quantification of number of green fluorescence-positive cells. (E) Immunofluorescence analysis of p65 (green staining; DAPI, blue staining). Objective magnification, x20. ZO, zonula occludens; Tβ4, thymosin β4; hBMVECs, human brain microvascular endothelial cells; LPS, lipopolysaccharide. *P<0.05 vs. untreated cells (control). #P<0.05 vs. LPS treated cells.