Extended Data Fig. 8: Combinatorial treatment strategies for HSV-1- driven neuroinflammation.

a, Exemplary images of HSV-1 GFP virus expression in sectioned and anti-GFP antibody immunostained organoids. Uninfected (CTRL), infected (HSV-1), ACV treated organoids (ACV), ACV and CDDO-Me co-treated (ACV+CDDO-Me) and ACV and NEC-1 cotreated (ACV+NEC-1). Lower panel: merged images, GFP (white), DAPI (blue). Scale bar - 500 µm. Representative image of three independent experiments with two iPSC lines (n=3 biological replicates/condition/iPSC line). b, Scatterplots showing all comparisons between viral GFP RNA, measured as Delta Ct with respect to the human DICER1 gene in cDNA from whole organoids lysate, viral GFP gDNA, measured as before in genomic DNA from the same organoids, and viral GP protein, measured from Western blot densitometric analysis, normalized on GAPDH. All correlation coefficients (Pearson) are shown in the top left of each plot. Sample identity is indicated next to each dot, representing the mean between three biological replicates (n=3, biological replicates). c, Exemplary images of ZO-1 and HSV-1 GFP virus expression in sectioned and immunostained organoids, infected with high (MOI=1) and low (MOI=0,2) multiplicity of infection (MOI) of HSV-1. Lower panel: merged images, ZO-1(red), GFP (white), DAPI (blue). Scale bar - 100 µm. Representative image of three independent experiments with two iPSC lines. d, e, ZO-1 fluorescent signal quantification in sectioned and immunostained organoids infected with high (d) or low (e) multiplicity of infection (MOI) of HSV-1, each dot represents one neuroepithelial loop, colors indicated cell lines: iPSC line 1 (dark blue), iPSC line 2 (bright blue). Lower panel, p-values for the fluorescent signal quantification f, g HSV-1-GFP signal quantification in immunostained organoids infected with high (f) or low (g) multiplicity of infection (MOI) of HSV-1 virus, each dot represents one neuroepithelial loop, colors indicated cell lines. Lower panel: p-values (Benjamini-Hochberg-corrected pairwise Wilcoxon Mann Whitney test for the fluorescent signal quantification, n=14,34,32,17,23 individual neuroepithelial loops quantified from 3,5,6,4,4 individual organoids per condition for panels d and f, n=43,31,33,22,21,38,29 loops from 5,3,3,2,3,3,3 organoids for panels e and g, from two independent hiPSC lines and two MOIs. Boxplot show lower quartile, median and upper quartile, whiskers represent upper and lower quartile ± the smallest value within 1.5-fold of the interquartile range respectively.

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