Extended Data Fig. 8: Characterization of neural progenitor cells and TBEV infection of neuronal cell cultures differentiated for 60 days.
a, Neural progenitor cells (NPCs) were immunostained for SOX2, NESTIN, GFAP, OLIG2, and PDGFRα and their cellular distribution was visualized by fluorescence microscopy. The number of NPCs expressing the indicated marker proteins was quantified manually or using ImageJ and a custom analysis pipeline in CellProfiler. Pooled data from one experiment is shown (n ≥ 400 individual cells per marker protein). Arrowheads indicate GFAP+ cells. Scale bar, 100 μm. b, Neural cells were differentiated in culture for 60 days, immunostained for the neuronal markers NeuN, MAP2, PSD-95, βIII-tubulin, LRP8 and GFAP, and their cellular distribution was visualized by fluorescence microscopy. Arrowheads indicate GFAP+ cells. Representative images of one experiment per condition are shown. Scale bar, 100 μm. c, The number of neural cell bodies (6.1 μm extension from the perimeter of the nuclei) from cells characterized in b and co-expressing LRP8 and βIII-tubulin or LRP8 and GFAP, respectively was quantified using ImageJ and a custom analysis pipeline in CellProfiler. Pooled data from one experiment is shown (n = 6000 individual cells per protein combination). d (left panel), Infection of neuronal cells characterized in b with TBEVSofjin (MOI, 0.02 IU) after pre-incubation with LRP8-LA1-2-Fc or human IgG (400 nM). Cells were subjected to immunostaining followed by immunofluorescence microscopy. Images from one experiment representative of two independent experiments are shown. Scale bar, 100 μm. (Right panel), TBEV-infected neural cell bodies in fluorescent images were enumerated using ImageJ and a custom analysis pipeline in CellProfiler. Pooled data from two independent experiments is shown as averages ± s.d. [n = 29 (human IgG) and n = 23 (LRP8-LA1-2-Fc), in which n represents fluorescent images]. Comparisons are presented between cells infected with human IgG- or LRP8-LA1-2-Fc-treated TBEV, respectively; data was analysed by two-tailed unpaired t-test: ****, P < 0.0001.
