Fig 2: Dpy30 directly regulates NPC differentiation and induction of key genes for differentiation. (A and B) Differentiation of NE-4C mouse NPCs. n = 3 biological repeats. (D–F) Differentiation of ReNcell VM human NPCs. n = 3 biological repeats. (A and D) Number of neurons and astrocytes (indicated) found in the control and KD culture after 9 days of differentiation. For A, a percentage plot was not possible due to extensively overlapping DAPI-positive cells in control (Scr) culture. As no Tuj1-positive cells were seen in KD culture, the conclusion based on cell number would be in agreement with percent cell calculations. Scr, scrambled control shRNA. Neurons were identified based on Tuj1 (for mouse) or NEUN (for human) staining, and astrocytes based on GFAP staining. (E) Percent astrocytes and neurons found in Scr and DPY30-KD ReNcell VM human NPCs after 9 days of differentiation. Astrocytes were identified based on GFAP staining. Neurons were identified based on long (>20 µm) fibers or NEUN staining. The plots show the percentage of the number of cells with indicated characteristics in the total number of DAPI-positive cells. UE, underestimated, due to extensively intertwined structure. (B and F) Relative mRNA levels were determined by qPCR and normalized to Gapdh/GAPDH for indicated genes upon Dpy30/DPY30 KD with and without treatment of differentiation conditions for 9 days in NE-4C (B) or ReNcell VM (F) cells. One of the biological repeats for ‘Scr Differentiated’ was set to one for each gene. Gene names are shaded in aqua for gliogenesis and in orange for neurogenesis. (C and G) Dpy30 and H3K4me3 ChIP-qPCR in NE-4C (C) or ReNcell VM (G) cells. Relative enrichment was generated by calculating percent input for each locus followed by normalizing to the percent input for the Map2/MAP2 locus in the control cells. Data represent the mean ± SD. Two factor ANOVA analysis followed with post hoc t-test was performed in B and F. ?P < 0.05, ??P < 0.01, and ???P < 0.001, by two-tailed Student’s t-test for the rest of the figure.

Fig 3: Dpy30 deficiency impairs neurogenesis and gliogenesis in the postnatal brain. (A and B) GSEA for RNA-seq results from hippocampus or SVZ cells (indicated) of P12 control and KO mice (n = 3 mice each) using indicated gene sets. NES, normalized enrichment score. (C–G) Staining for DAPI and Blbp (C), Dcx (D), NeuN (E), Gfap (F), Ki-67 (G) in coronal sections from P12 control and KO mice. Representative of three mice each genotype. (H) Quantifications of positive stained cells or area of the images in C–G. Both top and bottom subgranular zone/granular zone layers of the dentate were counted for control and the whole dentate (absence of a subgranular zone) was counted for KO. For SVZ, ImageJ software’s ‘area count’ was used to obtain area of positive staining and total area of SVZ. n = 9 (three mice, each with three different bregma levels). Data represent the mean ± SD in H. P-values are either labeled on top of the bars or ?P < 0.05, ??P < 0.01, and ???P < 0.001, by two-tailed Student’s t-test.

Fig 4: Lamin B1 accumulation induces the expression of markers of reactive astrocytes: The expression levels of reactive astrocytes markers were evaluated in U87-MG astrocyte-like cells transduced with Lamin B1-coding vector (ovLMNB1) and compared to wild type (WT) and mock-transduced cells (GFP). (A,B) display immunofluorescence staining of GFAP and vimentin, respectively, at 40× magnification (bar: 20 µM). Nuclei were stained using Hoechst 33342 (blue). Results are representative of at least five different fields. (A,B): In WT and GFP samples, only background fluorescence is seen for GFAP and vimentin, while the signal is strong in ovLMNB1 samples. (C): GFAP, vimentin, nuclear NF-kB, and c-Fos expression was measured in wild type, mock-transduced (GFP), and Lamin B1-overexpressing cells. Samples were normalized by using total protein normalization vs. wild type cells. Results are representative of three independent experiments. (D): Quantitative analysis of protein expression; wild type cells (WT) were used as reference sample. ** denotes p < 0.01 vs. corresponding mock- transduced sample (GFP).

Fig 5: High expression of SOCS3 in activated A1 astrocytes inhibited oligodendrocyte formation. (A) Schematic diagram showing the collection of astrocytes treated with control siRNA or SOCS3 siRNA, followed by culturing astrocytes with OPCs in the presence or the absence of IL-17A. (B) Representative images of OPCs cultured with astrocytes treated with control siRNA or SOCS3 siRNA for 72 h, which were immunostained for MBP and GFAP. Scale bar = 75 µm. (C) Percentage of MBP+ cells to total cell numbers. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA and Tukey’s test. Data in each statistical graph are presented as the mean ± SEM.