Fig. 4

TLR2 and TLR4 loss influence the spontaneous differentiation of in vitro-expanded spinal cord NPCs. A, B Representative immunofluorescence images of β3tubulin (neural marker; green) and GFAP (astrocytic marker, red) and the corresponding quantification data of the percentage of positive cells (lower panels) in NPCs grown in growth medium (A) or in differentiation medium (B) as summarized in the diagrams (top images). DAPI used for nuclei counterstaining (blue). C Morphological classification of the three distinct types of neurons identified from β3tubulin staining—type 1 (pyramidal-like cells; upper panel), Type 2 (rounded, with no cell expansions; central panel), and Type 3 (bipolar cells; lower panel). Quantification and comparative analysis of the percentage of the corresponding type of neurons shown for WT, TLR2^−/−, and TLR4^−/− NPCs; D Gene expression analysis of Dcx (early neuronal marker, upper graph) and MAP2 (late neuronal marker, lower graph). E Gene expression analysis of Neurogenin1 in growth medium (left) or differentiation medium (right) in WT, TLR2^−/− and TLR4^−/− NPCs. F (lower graph) Quantification and comparative analysis versus WT NPCs of the number of cells expressing higher GFAP protein expression levels in TLR2^−/− and TLR4^−/− cells; (upper panels) Binarized representative images for TLR2^−/− and TLR4^−/− NPCs showing cells with a fluorescence intensity above the WT levels threshold. G Protein expression levels of STAT3 in WT, TLR2^−/− and TLR4^−/− NPCs (α-tubulin as a loading control). H (Left) Representative images of immunofluorescence staining of Olig2 (orange) in NPC in growth medium (upper panels, day 1) and differentiation medium (lower panels, day 7). DAPI used for nuclei counterstaining. (Right) Quantitative analysis of Olig2 positive cells at one day (upper graph) and seven days (lower graph). I Gene expression analysis for NG2 (upper graph) and SOX10 (lower graph). Data shown as mean ± SEM. Results were assessed for normality using the Shapiro–Wilk test and one-way ANOVA with Tukey post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001