Fig. 4

Dietary inulin supplementation altered the proportion of astrocyte subpopulations in brainstem. A UMAP of all cell types in the brainstem of Ctrl_AD and Inulin_AD mice. B Violin plot of marker gene expression level of all cell types. C Cellular composition comparison between the Ctrl_AD and Inulin_AD groups. Western blots and quantification of NeuN (D) and MBP (E) levels in the brainstem homogenates of Ctrl_AD and Inulin_AD mice. Each dot represents one animal. Unpaired t-test was used to determine statistical significance. Data were presented as mean ± SD. n = 5 for both groups. F UMAP of astrocyte subtypes in the brainstem of Ctrl_AD and Inulin_AD mice. G Violin plot of marker gene expression level in astrocyte subtypes. H Comparison of astrocyte composition. Each dot represents one animal. Two-way ANOVA with Sidak’s multiple comparisons test was used to determine statistical significance. Data were presented as mean ± SD. I Volcano plot of DEGs of cluster 0 astrocytes against cluster 2. Red dots represent the genes that were upregulated in cluster 0 compared to cluster 2. Blue dots represent the genes that were downregulated in cluster 0. Absolute log2 fold change greater than 0.3 and P < 0.05 were used as selection criteria. J GO analysis of DEGs in astrocyte cluster 0 and 2. Pathways that were upregulated in cluster 0 compared to cluster 2 were shown in red. Pathways that were downregulated in cluster 0 were shown in blue. K Analysis of transcriptional trajectories in cluster 0 and 2 astrocytes. Arrows represent the predicted transcriptional flow based on RNA velocity analysis of spliced versus un-spliced mRNA transcripts. Cells at the border between cluster 0 and 2 were highlighted by the blue dashed circle. Heat map illustrating the latent splice time of cluster 0 and 2 astrocytes, based on splicing kinetics derived from the RNA velocity model. Color of each dot represents the level of progression of each cell along the differentiation trajectories defined by the RNA velocity model. Supporting data values for D–E were included in Additional file 6: Supporting data values. Original images of D–E were included in Additional file 7: original images