EMBO/SourceData huggingface dataset:
Figure 3Enrichment of H3K27me3 at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells.B. Enrichment of RNA polymerase II at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells. C. NDIME interacts with EZH2 (P = 0.0043) and SUZ12 (P = 0.0003), but not EED (P = 0.6720) and EZH1 (P = 0.0951) compared with IgG, as determined by RIP using EZH2, SUZ12, EED, and EZH1 antibodies at day 5 of neural differentiation. Nuclear RNA U1 as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).  NDIME levels following EZH2-RIP (P = 0.0028) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).  E. NDIME levels following SUZ12-RIP (P = 0.0015) in HEK293T cells compared with pcDNA3.1. Nuclear RNA U1 was used as a negative control. The presence of NDIME and U1 was measured by qRT-PCR with or without reverse transcriptase (RT).  F, Enrichment of EZH2 at the Mef2c promoter in NDIME-knockout cells at day 5 of neural differentiation compared with control cells.  G. Enrichment of SUZ12 at the Mef2c promoter in NDIME-knockout cells at day 5 of neural differentiation compared with control cells.  H. Expression of neural-lineage genes at day 5 of neural differentiation in the NDIME-knockout cells after adding the EZH2 inhibitor GSK126 (7.5 µM) partially rescued neural differentiation defects, as shown by qRT-PCR; the wild-type and NDIME-knockout cells treated with DMSO.
Figure 4 B. RIP analysis using EZH2 antibody for the co-transfection of Flag-EZH2 and NDIME exons 1-3 (P = 0.0177) or exon 4 (P = 0.4335) in HEK293T extracts compared with IgG. C. Verification of the efficiency of overexpression of exons 1-3 or exon 4 of NDIME in NDIME-knockout cells at day 5 of neural differentiation via qRT-PCR. Three different primers P1, P2, and P3 were used to analyze NDIME or exons of NDIME expression in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells (compared with control cells). The position of primers P1, P2, and P3 are shown in the schematic diagram. FACS , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation.  E. , expression of neural-lineage genes , in Ctrl, NDIME-/-, NDIME-/-+Rs26-Exon123, NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation.  F. Microscopy of the control, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. Scale bars, 100 µm.  G. NESTIN and GFP immunostaining of the cells in (F) at day 5 of neural differentiation. Scale bars, 100 µm. H. Enrichment of H3K27me3 at the Mef2c promoter of the cells in (F) at day 5 of neural differentiation.I. Western blot analysis of expression of MEF2C in the cells in (F) at day 5 of neural differentiation.
Figure 5A. Summary of the DEGs in the NDIME-knockout cells compared with control cells at day 5 of neural differentiation.C. Summary of the DEGs in the MEF2C-knockdown cells compared with control cells at day 5 of neural differentiation.I. Heatmap illustrating the expression of selected neurectoderm genes and mESCs-specific genes that were shown as log2 FPKM in Ctrl, NDIME-/- , shCtrl and shMef2c mESCs at day 5 of neural differentiation. Each lane corresponds to an independent biological sample. J. qRT-PCR analysis of representative co-upregulated and co-downregulated genes in (G) at day 5 of neural differentiation.  L. Relative expression of selectively down-regulated ASD risk genes from the DEGs of the NDIME-knockout cells compared with control cells at day 5 and from the DEGs of the MEF2C-knockdown cells compared with control cells at day 5.
Figure 6 A. qRT-PCR analysis of NDIME (P = 0.0051) and MEF2C (P = 0.0062) in VPA-treated mice and saline-treated mice; n = 9 per group.  B. Enrichment of EZH2 at the Mef2c promoter in VPA-treated mice and saline-treated mice (compared with saline-treated mice); n = 9 per group. C. Western blot analysis of the protein levels of MEF2C and GAPDH in the hippocampus of VPA-treated mice (n = 9) and saline-treated mice (n = 10). D. Immunofluorescence images for MEF2C in the hippocampus of VPA-treated mice and saline-treated mice. Nuclei were stained with Hoechst 33342 (blue); n = 9 per group. Scale bars, 50 µm. E. Statistical analysis of the grayscale levels of MEF2C in (C) by ImageJ (P = 0.0074); saline-treated mice (n = 10), VPA-treated mice (n = 9). F. Statistical analysis of the Mean MEF2C staining intensity/unit area in (D) by ImageJ (P = 0.0096); n = 9 per group.  G. Immunofluorescence images for GFP in the hippocampus of saline-treated mice, VPA-treated mice, and VPA-treated mice with viral-mediated expression of NDIME H. Immunofluorescence images for MEF2C of mice in (G); n = 9 per group. Scale bars, 50 µm. I-K. Adeno-associated virus overexpression of NDIME in the dentate gyrus (DG) of the dorsal hippocampus of VPA-treated mice rescued autism-like social deficits. Open field test (I), three-chamber test (J), and elevated-plus maze (EPM) test (K). Data in (I-K) are represented as mean ± SEM; n = 9 per group. Asterisks indicate a difference between VPA+AAV-GFP and Saline+AAV-GFP, whereas hash tags indicate a difference between VPA+AAV-NDIME-GFP and VPA+AAV-GFP. #P < 0.05, **/##P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
Figure 1C) Western Blot showing the levels of the PgP and control (α-TUBB) in RPE-1 parental and Taxol resistant clones.E) Representative smRNA-FISH images of RPE-1 Parental and clones for the ABCB1 gene and DAPI. The images are projections of 0,5μm sections and a total 5 μm in thickness. Scale bar, 15μm.
Figure 2A) TLA analysis for ABCB1 contacts in RPE-1 parental and Taxol resistant clone sg6C9 covering the whole genome. Green arrow in sg6C9 shows a de novo interaction found between ABCB1 and a region in chromosome 6. B) TLA analysis for RPE-1 Parental and sg6C9. Zoom in in the region of chr6 with de novo interaction for sg6C9. Image modified from IGV viewer.D) PCR products using the primers in C(1) over the ABCB1 and EEF1A1 regions in RPE-1 Parental and the different Taxol resistant clones. E) PCR products using the primers in C(2) over the specific break site in the ABCB1 promoter in RPE-1 Parental and the different Taxol resistant clones.G) Percentage of RPE-1 cells targeted with the ABCB1 promoter sgRNAs found with vector integration. Percentages were calculated from data in Fig 1A. Each dot represents a CFA replicate experiment. Horizontal bars represent the mean of lentiviral integratation per sgRNA.
Figure 3A) H3K9me3 ChIP-sequencing tracks from the q21.12 arm of chromosome 7 in RPE-1 cells (Parental and Taxol-resistant clones). The ABCB1 gene region is shown in pink. Each colored track shows the H3k9me3 profile of a different Taxol-resistant clone.B) H3K9me3 ChIP-qPCR in the ABCB1 promoter region. Bar graph show primer pairs amplifying the sgRNA#6 break and spanning this region (+/- base pairs) for each Taxol-resistant clone compared to the parental. Bar plots show the mean of H3K9me3 relative enrichment. Each dot represents a technical replicate (n=3).
Figure 1VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (B) Cells were lysed 48h post-infection and viral protein as well as cell cycle associated protein expression was analysed by western blot. N, nucleocapsid.VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (C) VERO AT2 Cells were fixed 24h post-infection and stained for viral proteins and cyclins. Arrowheads highlight un-infected cells and cyclin D/A nuclear localization. Arrowheads: Nuclear cyclin staining in uninfected cells. Scale bars: 20μm. (D) VERO AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗∗p < 0.0001.(E) A549 AT2 cells were infected with WT, alpha (α), and delta (∆) SARS-CoV-2 variants. Cells were lysed 48h post-infection and viral, cyclin proteins expression was analysed by western blot. N, nucleocapsid.(F) A549 AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 2A549 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 18h post-infection. Cells were lysed 24h post-addition of inhibitor and cyclins and viral proteins were detected by western blot. (N) nucleocapsid. Densitometry analysis of western blots for D-cyclins (normalized to actin) in A549 AT2 cells. Plots are average of 2 (cyclin D1), 3 (cyclin D3) biological replicates. Bars indicate mean with SD. Statistical analysis was performed using ordinary two way ANOVA; ns, non-significant; ∗p < 0.1.A459 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 8h post-infection. Cells were fixed and stained 24h post-addition of inhibitor. (N) nucleocapsid.Scale bars: 40μm. Contour lines represent outlines of infected cells.Quantification of D3 cyclin relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells have been counted. Bars indicate mean with SD. Statistical analysis was performed using ordinary two-way ANOVA; ∗∗∗p < 0.001.
Figure 3(A, D and A-cyclins were depleted using siRNA in A549 AT2 cells. (A, Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.B) D and A-cyclins were depleted using siRNA in A549 AT2 cells. Cells were infected 18h later with Delta (∆), Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.001, 0.1, 0.1 respectively. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for TCID50 (B). (B, Virus titres in cell culture supernatants were determined as TCID50 Data information: Graphs represent average of n=3 (B, Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.(C, D and A-cyclins were depleted using siRNA in VERO AT2 cells. C) Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.D) D and A-cyclins were depleted using siRNA in VERO AT2 cells. Cells were infected 18h later with Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.1. Cells were washed 4h post-infection and new media added. Supernatants and cells were collected at 24h and 48h later. D) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells. Data information: Graphs represent average of n=3 D); Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.Cyclin D3 was depleted using siRNA in Calu3 cells. Cells were infected 48h later with Delta SARS-CoV-2 at MOI 0.01. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for western blot and TCID50. Uninfected cell lysates were used for western blot. Data information: Graphs represent average of n=4 (E) biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.
Figure 5A549 AT2 cells were depleted for D and A2 cyclins (A) A representative western blot from lysates of uninfected knock-down cells.(E) A549 AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and VSV-G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 (mutant not degraded by proteasome). Cells were infected 24h later and collected 48h post-infection for flow cytometry analysis of early S and western blot. n=2 biological replicates; Ordinary two-way ANOVA with Sidak's multiple comparisons test: ns, non-significant; ∗∗p < 0.01. Bars indicate mean with SD.(F,G) VERO AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS-CoV-2. Cells were fixed and stained for D-cyclins 24h later. (F) Example of acquisition using automated microscopic platform. Cells are identified for infection, expression of cyclin D3, and cell cycle (Red/arrow=G1phase; Green/arrowhead=S/G2/M; Red+Green/arrowhead=early S). Scale bar: 20μm. (G) Quantification of D-cyclins re-localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony (PerkinElmer). At least 50-200 cells were analysed in each condition. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ∗p < 0.1.
Figure 6(A) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E or nsp9, and control plasmid (EV). Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA, and cyclin D3 antibodies.(B) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E, M, both E and M or nsp9. Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA antibodies. WCL, whole cell lysate.(C-G) 293T GFP11 cells were transfected with Spike, and/or with Envelope, Membrane, and cyclin D3. 24h post-transfection cells were seeded at a 1:1 ratio with Vero-GFP10 cells and percentage of GFP+ve area (syncytia) were determined 18h later. S, Spike; E, Envelope; M, Membrane; D3, cyclin D3. (C,D) Representative images of GFP+ syncytia. Scale bars: 40μm. (E-G) Quantification of cell-to-cell fusion showing percentage of the GFP+ve area to the acquired total cell area. (E) n=4 biological replicates. (F,G) n = 4 biological replicates; technical triplicates shown; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01 . Bars indicate mean with SD.(I) Representative example of western blot from purified SARS-CoV-2 Virus Like Particles (VLPs) and whole cell lysates (WCL). MCM2 was used as loading control. (J) Quantification of protein incorporation (into VLPs) or expression (in WCL). n = 4 biological replicates; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗p < 0.001. Bars indicate mean with SD. N, nucleocapsid; M, membrane; E, envelope; S, spike.
Figure 1(B-C) Western blot analysis (B) and densitometric quantification (C) of OTUD1 in aortas of control and Ang II-induced mice. β-actin was used as the loading control (n=5 biological replicates). (D) mRNA levels of OTUD1 in aortas of control and Ang II-induced mice were determined by RT-qPCR. The values were normalized to Rn18s (n=5 biological replicates).(E) Representative immunofluorescence staining of CD31 (green) and OTUD1 (red) of aortic sections (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and OTUD1 (red) of aortic sections (scale bar=25 μm).(G-H) Time-course of OTUD1 induction in response to Ang II in HUVECs. HUVECs were exposed to 1 μM Ang II for indicated time. Western blot analysis (G) and densitometric quantification (H) of OTUD1 were shown (n = 3 biological replicates). GAPDH was used as the loading control.
Figure 2(A) Representative images of H&E staining of aortic sections of the mice from four groups (scale bar=25 μm). E, endothelium; M, media; A, adventitia. (B) The quantification of arterial wall thickness in panel A; n=5 biological replicates. (C) Representative images of Masson's Trichrome staining of aortic sections of the mice (scale bar=50 μm). (D) The quantification of collagen deposition in panel C; n=5 biological replicates.(E-F) Western blot analysis (E) of VE-cadherin, Vimentin, Twist, Snail, and OTUD1 and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues of the mice. β-actin was used as the loading control (n=5 biological replicates).(G-H) mRNA levels of Cdh5, Vim, Twist1, Snai1 (G), Tgfb1 and Col1a1 (H) in aortic tissues of the mice were examined by real-time qPCR assay. The values were normalized to Rn18s (n=5 biological replicates).
Figure 3(B) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.(E) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.
Figure 4(C) Co-immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag-OTUD1. Exogenous Flag-OTUD1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G.(G) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (H) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and Flag-OTUD1 or Flag-OTUD1(C320S), and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (I) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, Flag-OTUD1, and HA-Ub, HA-Ub-K48 (K48 only), or HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody.
Figure 5(A-B) 293T cells were transfected with various amount of Flag-OTUD1. Western blot analysis (A) and densitometric quantification (B) of SMAD3. GAPDH was used as the loading control (n=3 biological replicates).(E-F) Western blot analysis (E) and densitometric quantification (F) of SMAD3 in aortic tissues of the mice from four groups. β-actin was used as the loading control (n=5 biological replicates).(G) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody.(H) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3 and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. IgG, immunoglobulin G.
Figure 6(A-B) Representative images (A) of H&E staining of aortic sections (scale bar=25 μm) and quantification of arterial wall thickness (B); n=5 biological replicates. E, endothelium; M, media; A, adventitia. (C-D) Representative images (C) of Masson's Trichrome staining of aortic sections (scale bar=25 μm) and quantification of collagen deposition (D); n=5 biological replicates.(E-F) Western blot analysis (E) and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues. β-actin was used as the loading control (n=5 biological replicates).
Figure 1Immunostaining for CCN1 (green) in the adult brain coronal section. Scale bar: 50 μm. The insert refers to the same image with DAPI staining to show cell nuclei (blue). LV, lateral ventricle.Adult V-SVZ whole-mounts stained for CCN1 (white) and S100β (green). Scale bar: 10 μm.Adult V-SVZ whole-mounts stained for CCN1 (white), GFAP (green) and VCAM1 (magenta). Arrowhead points to a VCAM1+ B1 cell surrounded by CCN1-expressing ependymal cells. Scale bar: 10 μm.Confocal images tracing a VCAM1+GFAP+ cell reveal CCN1 expression is absent in B1 cells. Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit. White arrowheads point to the process of the B1 cell. Scale bar: 10 μm.
Figure 2V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm.V-SVZ whole-mounts stained for γ-tubulin (cyan) and GFAP (red). Arrowheads point to B1 cells in the pinwheel center. Scale bars: 500 μm (left), 50 μm (middle), 10 μm (right).Densities (cells/mm2) of B1 cells in the adult V-SVZ. n=4, ***p=0.0005.V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ. n=4, **p=0.002. Densities (units/mm2) of NSC units in the adult V-SVZ. n=4, **p=0.0022. Distribution of NSC units with different numbers of B1 cells. n=4, ****p<0.0001 (1 B1 cell/unit), **p=0.0025 (2 B1 cells/unit), **p=0.0063 (3 B1 cells/unit), *p=0.0385 (4 B1 cells/unit).
Figure 3Densities (cells/mm2) of B1 cells in the aged V-SVZ. n=3, **p=0.0099.Densities (cells/mm2) of VCAM1+ B1 cells in the aged V-SVZ. n=3, **p=0.0041.V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white) in aged mice. Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).Densities (units/mm2) of NSC units in the aged V-SVZ. n=3, **p=0.0093. Average numbers of B1 cells in a NSC unit. n=3, n.s. not significant.
Figure 4Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 2 days after TAM treatment. n=4, *p=0.0141. V-SVZ whole-mounts stained for GFAP (red) and Ki67 (cyan). Arrowheads point to dividing GFAP+ cells. Scale bar: 10 μm.Numbers of ASCL1+ cells in V-SVZ coronal sections 2 days after TAM treatment. n=3, **p= 0.0087. V-SVZ coronal sections stained for ASCL1 (red) and nuclei (blue). Scale bar: 50 μm.Densities (cells/mm2) of BrdU+ cells in the OB granule cell layer (GCL) 2 days after TAM treatment. n=4 (Ctrl) and 3 (Ccn1cKO), **p=0.0086. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 4 weeks after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.Numbers of ASCL1+ cells in V-SVZ coronal sections 4 weeks after TAM treatment. n=3, n.s. not significant. Scale bar: 20 μm.Densities (cells/mm2) of BrdU+ cells in the OB GCL 4 weeks after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 16 months after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.