Fig. 6: Exogenous p-tau induces phosphorylation for essential antiviral activity.
ReNcell VM cultures were infected with HSV-1 to determine the degree of immune response. a, One-week-old 2D ReNcell VM uninfected and infected cultures were immunoprobed with L-azidohomoalanine (AHA) and anti-p-tau (PHF1) and then labeled with fluorescent secondary antibodies and analyzed for neurons (GFP), p-tau (594) and AHA (647) fluorescence by confocal microscopy. b, Distribution plot of individual AHA and p-tau pixel intensities after infection. c, Pearson’s correlation coefficient (error bars represent 95% confidence interval) of images of internalized AHA and PHF1 in ReNcell VM cells. d, Fluorescent image captures from nine wells over three experiments were compared by GA3 software in Nikon Elements for p-tau fluorescence (****P < 0.0001) between azide-positive and azide-negative cells. e, One-week-old 2D ReNcell VM cultures were preincubated with dilutions of a GSK-3β inhibitor (scale of 50 nM to 25 nM) for 24 hours followed by a 24-hour HSV-1 infection and imaged by confocal microscopy for red fluorescence. Whole-well images were analyzed using Nikon Elements GA3 to compare the number of HSV-1 plaques between conditions (F3, 53 = 2.806, *P = 0.0268). f,h, Conditioned media collected from 1-week-old ReNcell VM uninfected (f) and 1-hour HSV-1 infected (h) (F2, 2 = 23.85, P** = 0.0025) cultures pretreated with 2N4R GSK-3β p-tau (1.25 µg ml−1) were run on a proinflammatory cytokine MSD. g, One-week-old 2D ReNcell VM cultures were preincubated with 2N4R GSK-3β p-tau (1.25 µg ml−1) and anti-IFNγ antibody (10 µg ml−1) for 24 hours followed by a 24-hour HSV-1 infection and imaged by confocal microscopy for red fluorescence. Whole-well images were analyzed using Nikon Elements GA3 to compare the number of HSV-1 plaques between conditions (F3, 56 = 4.863, *P = 0.0386). Box plots are representative of ±s.e.m. ((e, n = 11), (f, n = 6), (g, n = 15), (h, n = 3)) depicting median and interquartile range, with whiskers denoting variability according to Tukey’s method. Statistical mean comparisons were calculated by two-tailed unpaired Welch’s t-test (d), one-way ANOVA using Dunnett’s multiple comparisons test (e), two-way ANOVA using Tukey’s multiple comparisons test (f), one-way ANOVA using Tukey’s multiple comparisons test (g) and two-tailed unpaired t-test (h).
