Fig. 6: 4-PBA induced alteration of Cx43 (expressed by Gja1 mRNA) alters LACV induced vascular leakage and viral entry into weanling mice brain.
From: Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis
a, b LACV-infected (10 MOI) bEnd.3 cells, were treated with 4-PBA (0–10 mM) and at different hpi, imaged to measure (a) viral intensity or virus-induced cell depletion (N = 4 samples, data collected based on 25 images obtained from each sample and mean with SD is shown) or (b) localization of Cx43 (red), LACV (green) and Hoechst (blue) (representative of ~12–25 images obtained each condition, P = 0.1142, < 0.0001 and 0.0097 from PBA 2.5, 5and 10 mM, compared to PBA 0 mM). White arrows represent punctate staining of Cx43. c Western blot and densitometric analyses of Cx43 protein levels in 4-PBA treated bEnd.3 cells. Gapdh is a loading control and normalization factor for quantitation (N = 3 each condition and mean with SD is shown, P = 0.7647, 0.0043 and 0.4589 for PBA 2.5, 5 and 10 mM those are compared to PBA 0 mM). d Weanling mice, infected with 2000 PFU of LACV, were treated with vehicle (LV) or 500 mg/kg-day 4-PBA (LP) until 5 dpi and assessed for neurologic endpoint (N = 9 mice for each condition, P = 0.0074). e Effect of 4-PBA treatment on the correlation between LACV and Gja1 expression in mouse brains at 3 dpi (N = 9 LV and N = 11 LP brain RNA samples obtained from mice, P = 0.0311 and 0.0147 for LACV and Gja1 comparison). f Effect of 4-PBA on entry and localization of LACV as assessed by confocal imaging of mouse brain slices at 4 dpi (red: ZO1, green: LACV, blue: Hoechst/nuclei). (a) two-way ANOVA followed by Dunnett’s test and all groups were compared with vehicle for same time pi, (b) one-way ANOVA followed by Dunnett’s test, individual points represent separate imaging fields, (c) multiple, two-tailed unpaired t-tests, d Mantel-Cox log rank test and e, two-tailed unpaired t-test where *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 and N = 3–4 for each experiment. Scale bar = 100 um (white, for regular images), scale bar = 10 um (yellow, for zoomed in images).
