Fig. 2

Modulation of 3-O sulfation results in decreased uptake of tau-AF488 in HCT-116 cells. a Using live-cell microscopy to measure tau-AF488 uptake, it is possible to observe a reduction in the number and intensity of tau-AF488 puncta. b Quantification of tau-AF488 uptake normalized to the uptake of WT cells, shows a significant reduction of 50% in tau seeds uptake by the HS3ST1^−/− cells (two-way ANOVA with Sidak’s post hoc against WT cells treated with tau seeds, **** p < 0.0001) (c) Live-cell microscopy of HS3ST1^−/− cells transfected with either HS3ST1-mKate2 WT construct or inactive mutant forms of HS3ST1, shows an increase in the number and intensity of tau-AF488 seeds puncta in HS3ST1 overexpression conditions. The constructs were designed to express mKate2, a fluorescent protein, at the C-terminal to allow the script to quantify the uptake of tau PFFs in transfect cells. The bottom panel shows the images without the mKate2 signal for easier visualization of the tau PFFs puncta (d) Quantification of tau-AF488 seeds uptake, normalized to the maximum uptake in WT cells reveals no significant difference in Tau uptake levels between HS3ST1^−/− cells overexpressing HS3ST1 and WT cells mock transfected, indicating a rescue of uptake upon HS3ST1 overexpression (two-way ANOVA with Sidak’s post hoc against WT cells mock transfected, n.s p > 0.9999). e Using live-cell microscopy to measure tau-AF488 uptake in HCT-116 WT cells, it is possible to observe a reduction in the number and intensity of tau-AF488 puncta upon pre-treatment of cells with ATIII protein (f) Quantification of tau-AF488 seeds uptake by HCT-116 WT incubated with ATIII show a 60% reduction of tau uptake, which is not observed on HS3ST1^−/− cells incubate incubated with ATIII (two-way ANOVA with Tukey’s post hoc, **** p < 0.0001, n.s p = 0.37). Data represented as mean ± SD of 3 or 4 independent experiments (biological replicates). For images acquisition, 13 fields with 4 z-stacks were analyzed with around 2000 cells per field