cell nutrients 2025 17 1682 4 of 15 signaling  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Vitamin D regulates olfactory function via dual transcriptional and mTOR-dependent translational control of synaptic proteins

doi: 10.1101/2025.05.02.651619

Figure Lengend Snippet: A. Venn diagram showing example overlapping DEGs from snRNA-seq (comparing VDR□ Glu3 groups: VD- hyper vs. VD- hypo and VD- hypo vs. VDD) and spatial transcriptomics (comparing VDR□ spots: VD- hyper vs. VD- hypo and VD- hypo vs. VDD). Genes marked in pink represent DEGs confirmed by RNA sequencing in control vs. VDR knockdown (VDR-KD) mice; genes in purple correspond to DEPs from proteomic analysis (VD- hyper vs. VD- hypo or VD- hypo vs. VDD); genes in blue are confirmed by both techniques. B-C. Dot plots showing expression of translation-related DEGs in VDR□ Glu3 neurons from snRNA-seq ( B ) and VDR□ spots from spatial transcriptomics ( C ) across three groups (VD- hyper , VD- hypo , and VDD). Dot size represents the percentage of cells expressing the gene; color indicates averaged scaled expression levels. D-G. Relative expression levels of eIF5A ( D ), Rps24 ( E ), Rps27a ( F ), and eIF3B ( G ) in LC-MS-based proteomic analysis (n=4 mice per group; unpaired t-test). H-K. Levels of mTOR signaling components, including Rps6 ( H ), eIF4EBP1 and p-eIF4EBP1 ( I ), eIF4EBP2 ( J ), and eIF4E ( K ), in the OB of the three mouse groups, measured by wes (n=3 mice per group; one-way ANOVA with Tukey’s multiple comparisons). L-O. Levels of regulators of mTOR signaling ( L-M ), mTOR and p-mTOR ( N ), and downstream targets S6K and p-S6K ( O ) in the OB of the three mouse groups, measured by wes (n=3 mice per group; one-way ANOVA with Tukey’s multiple comparisons). P. Experimental schematic of whole OB synaptosome preparations and protein-level validation using quantitative wes . Q-U. Components of mTOR signaling, including MNK and p-MNK ( Q ), Rps6 ( R ), eIF4E ( S ), eIF4EBP1 and p-eIF4EBP1 ( T ), and eIF4EBP2 ( U ), in synaptosome preparations from the OB of three mouse groups, measured by wes (n=4 mice per group; one-way ANOVA with Tukey’s multiple comparisons). V-B’. Relative mRNA expression of Rpl4 ( V ), Rpl7 ( W ), Rpl21 ( X ), Rpl37 ( Y ), Rpl37a ( Z ), Rps13 ( A’ ), and Deptor ( B’ ) in control (Ctrl) and VDR-KD mice, analyzed by RNA sequencing (n=3 mice per group; statistical analysis conducted using standard pipelines for RNA-seq data, with normalization and multiple testing correction applied). Each symbol represents a biological replicate. Data are presented as mean ± SEM. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns indicates no statistical significance. FPKM: Fragments per kilobase million.

Article Snippet: The following antibodies were used: mouse anti-β-actin (1:200, Novus, NB600-501), rabbit anti-AKT (1:200, Cell Signaling Technology, 4691), rabbit anti-p-AKT (1:20, Cell Signaling Technology, 4060), rabbit anti-eIF4E (1:100, Cell Signaling Technology, 2067), rabbit anti-eIF4EBP1 (1:100, Cell Signaling Technology, 9644), rabbit anti-p-eIF4EBP2 (1:200, Cell Signaling Technology, 2855), Rabbit anti-eIF4EBP2 (1:20, CST, 2845), rabbit anti-GAPDH (1:5000, Cell Signaling Technology, 5174), mouse anti-gephyrin (1:4000, Synaptic Systems, 147111), rabbit anti-mGluR1 (1:500, Cell Signaling Technology, 12551), rabbit anti-MNK (1:100, Cell Signaling Technology, 2195), rabbit anti-p-MNK (1:20, Cell Signaling Technology, 2111), mouse anti-mTOR (1:1000, CST, 4517), rabbit anti-p-mTOR (1:50, CST, 5536), rabbit anti-PI3K (1:3000, Cell Signaling Technology, 4249), rabbit anti-p-PI3K (1:20, Cell Signaling Technology, 17366), rabbit anti-PSD95 (1:100, Cell Signaling Technology, 3409), rabbit anti-Rps6 (1:100, Cell Signaling Technology, 2217), rabbit anti-S6K (1:200, CST, 33475), rabbit anti-p-S6K (1:50, CST, 9234), mouse anti-synapsin1 (1:7000, Synaptic Systems, 106011), rabbit anti-VDR (1:500, Cell Signaling Technology, 12550), mouse anti-VGAT (1:20, Synaptic Systems, 131011), mouse anti-vGlut1 (1:7000, Synaptic Systems, 135011), rabbit anti-vGlut2 (1:100, Cell Signaling Technology, 16066).

Techniques: RNA Sequencing, Control, Knockdown, Expressing, Liquid Chromatography with Mass Spectroscopy, Biomarker Discovery