(A) Schematic diagram of experimental design. Created with BioRender. (B) Volcano plots showing upregulated or downregulated genes in mouse OPCs cultured on FBLN2-coated (10 μg/mL) wells for 6 hours, identified by RNA sequencing. (C) Top upregulated pathways predicted by IPA from DEGs. RNA-sequencing data were acquired from 3 replicates per group (PBS or FBLN2). (D) Representative images of mouse OPCs cultured on control and FBLN2-coated (10 μg/mL) wells with or without Notch inhibitor (SAHM1, 10 μM) after 24 hours (top) and 72 hours (bottom). (E and F) Quantification for mean process outgrowth (E) and number of O4⁺ cells after 24 hours (F) (n = 4 independent experiments). (G) Number of O4⁺MBP⁺ cells at 72 hours (n = 3 independent experiments). (H) Number of O4⁺ cells 24 hours after transfection with 2 Notch1 siRNAs (200 nM each; n = 3 independent experiments). Each experiment (dot) in E–H included 3–4 replicates; 1-way ANOVA, Tukey’s post hoc. (I) Schematic depicting the Notch pathway reporter assay using the constitutively expressing Renilla luciferase vector (positive control) and CSL (CBF1/RBP-Jκ) firefly luciferase reporter vector. (J) Bar graph comparing relative luciferase activity (firefly to Renilla) in HEK239 cells transfected with luciferase reporter or negative control vectors in the presence or absence of 10 μg/mL FBLN2 (n = 8–10 replicates over 2 separate experiments; 1-way ANOVA, Bonferroni post hoc). (K and L) Ridge plots comparing expression of select genes involved in Notch pathway across oligodendrocyte subclusters (0, DA-MOL; 1, MOL; 2, IFN-MOL; 3 and 4, Stressed-OL; 5, COP; 6, NFOL) (K) and experimental groups (L). (M) Immunofluorescence images of MS brain sample labeled for Olig2 and NICD. White arrows show the presence of Notch signaling in oligodendrocytes. Scale bars: 100 μm, insets, original magnification, 2×. Data are presented as mean ± SEM.