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Proteintech sin3a
Sin3a, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+sin3a/SIN3A+Antibody/pm41663400-400-56-57
Average 93 stars, based on 12 article reviews

sin3a - by Bioz Stars, 2026-09

93/100 stars

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Western Blot:

Article Title: SLC14A1 prevents oncometabolite accumulation and recruits HDAC1 to transrepress oncometabolite genes in urothelial carcinoma
Article Snippet: .. The protein complexes were eluted from antibody/beads after washing and subjected to immunoblot analysis by probing anti-SIN3A (Proteintech, USA), -HDAC1 (Cell Signalling, USA), -ARID4B (Proteintech) or -SUDS3 (Novus Biologicals, USA) antibody. .. pKM2L-phHKII (region #1, RDB05882, RIKEN, Renilla luciferase) and pGL4.54[luc2/TK] Vector (E5061, Promega, USA) were cotransfected into the mock - and SLC14A1-NLS -overexpressing J82 cells in a 96-well white plate by using PolyJetTM In Vitro DNA Transfection Reagent (Signagen®, USA).

Saline:

Article Title: Chitosan/PLGA-based tissue engineered nerve grafts with SKP-SC-EVs enhance sciatic nerve regeneration in dogs through miR-30b-5p-mediated regulation of axon growth
Article Snippet: Protein samples were mixed with 5 × SDS-PAGE loading buffer (Beyotime) and boiled for 10 min. A total of 20 μg of protein was separated using 10 % SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). .. Subsequently, the membranes were blocked in Tris buffered saline/Tween 20 (TBST) solution containing 5 % skim milk for 2 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: anti-Sin3a (14638-1-AP, Proteintech, 1:1000), anti-GAP43 (ab75810, Abcam, 1:1000), anti- p -STAT3 (9145, CST, 1:1000), anti-STAT3 (30835S, CST, 1:1000), anti- p -CREB (9198, CST, 1:1000), anti-CREB (9197, CST, 1:1000), anti- p -ERK1/2 (4370T, CST, 1:1000), anti-ERK1/2 (4695T, CST, 1:1000), anti- p -AKT (9272S, CST, 1:1000), anti-AKT (9271S, CST, 1:1000), anti- p -S6 (5364T, CST, 1:1000), anti-S6 (2217S, CST, 1:1000), anti-GAPDH (60004-1-Ig, Proteintech, 1:5000) and anti-β-actin (AC004, Abclonal, 1:10000). ..

Incubation:

Image Search Results

(A) External views (left) and H&E stained midsagittal sections (right) from control and Sin3A CKO mice at postnatal day 40 (P40), showing a reduction in cerebellar size and foliation. Scale bars indicate 250 μm. (B) Immunofluorescence staining for Cre recombinase at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Ki67 and NeuN at P7 in the cerebellum of control and Sin3A CKO mice. A series of sections from the lateral to medial cerebellum revealing significant variability in Sin3A CKO across different sagittal planes, attributable to disorganization within the tissue architecture. The dashed lines delineate the boundaries of different layers within the cerebellar cortex. EGL: external granular layer, ML: molecular layer, IGL: internal granular layer, PWM: prospective white matter. Arrows indicate acellular gaps in EGL. Arrowheads indicate ectopic clusters of GCPs. Scale bar indicates 250 μm. (D and E) Quantification of the cross-sectional area of EGL (D) and the number of Ki67 + cells (E) in EGL. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group. (F and G) Quantification of the cross-sectional area of IGL (F) and the number of NeuN + cells (G) in IGL. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group.

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) External views (left) and H&E stained midsagittal sections (right) from control and Sin3A CKO mice at postnatal day 40 (P40), showing a reduction in cerebellar size and foliation. Scale bars indicate 250 μm. (B) Immunofluorescence staining for Cre recombinase at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Ki67 and NeuN at P7 in the cerebellum of control and Sin3A CKO mice. A series of sections from the lateral to medial cerebellum revealing significant variability in Sin3A CKO across different sagittal planes, attributable to disorganization within the tissue architecture. The dashed lines delineate the boundaries of different layers within the cerebellar cortex. EGL: external granular layer, ML: molecular layer, IGL: internal granular layer, PWM: prospective white matter. Arrows indicate acellular gaps in EGL. Arrowheads indicate ectopic clusters of GCPs. Scale bar indicates 250 μm. (D and E) Quantification of the cross-sectional area of EGL (D) and the number of Ki67 + cells (E) in EGL. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group. (F and G) Quantification of the cross-sectional area of IGL (F) and the number of NeuN + cells (G) in IGL. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: Staining, Control, Immunofluorescence, Two Tailed Test

(A) Ki67 and EdU staining in cerebellum of control and Sin3A CKO mice at P0 and P7. Nuclei were counterstained with DAPI. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate cells positive for both Ki67 and EdU. Scale bars indicate 250 μm. (B) Quantification of the percentage of Ki67 + cells relative to the total number of DAPI + cells in EGL at P0 and P7. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, n.s. p>0.05. n= 3 mice per group. (C) Quantification of the percentage of EdU + /Ki67 + double-positive cells relative to the total number of Ki67 + cells in EGL at P0 and P7. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, n.s. p>0.05. n= 3 mice per group. (D) Immunofluorescence staining for p27 at P0 and NeuN at P7 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. The dashed lines delineate the outer margin of the EGL. Scale bars indicate 250 μm. (E) Quantitative analysis showing the percentage of p27 + or NeuN + cells relative to the total cell population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p ≤ 0.05, ** p ≤ 0.01. n= 3 mice per group. (F) Ki67, NeuN and EdU staining in the cerebellum of control and Sin3A CKO mice at P7. Nuclei were counterstained with DAPI. Mice were administrated EdU 48 hour before sacrifice. Arrowheads indicate cells positive for both NeuN and EdU, and arrows indicate cells positive for both Ki67 and EdU. The dashed lines delineate the boundaries of different layers within the cerebellar cortex. Scale bars indicate 250 μm. (G) Quantification of the percentage of EdU + /NeuN + double-positive cells relative to the total number of EdU + cells. Shown are mean ± SEM. Two-tailed t test: *** p ≤ 0.001. n= 3 mice per group. (H) Immunofluorescence analysis for Ki67 at P15 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bars indicate 250 μm. (I) Immunofluorescence staining for cleaved caspase-3 (CP3) and Ki67 at P0 and P7 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate CP3 + cells. Scale bars indicate 250 μm. (J) Quantification of the CP3 + cells in the EGL. Shown are mean ± SEM. Two-tailed t test: * p ≤ 0.05. n= 3 mice per group.

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Ki67 and EdU staining in cerebellum of control and Sin3A CKO mice at P0 and P7. Nuclei were counterstained with DAPI. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate cells positive for both Ki67 and EdU. Scale bars indicate 250 μm. (B) Quantification of the percentage of Ki67 + cells relative to the total number of DAPI + cells in EGL at P0 and P7. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, n.s. p>0.05. n= 3 mice per group. (C) Quantification of the percentage of EdU + /Ki67 + double-positive cells relative to the total number of Ki67 + cells in EGL at P0 and P7. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, n.s. p>0.05. n= 3 mice per group. (D) Immunofluorescence staining for p27 at P0 and NeuN at P7 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. The dashed lines delineate the outer margin of the EGL. Scale bars indicate 250 μm. (E) Quantitative analysis showing the percentage of p27 + or NeuN + cells relative to the total cell population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p ≤ 0.05, ** p ≤ 0.01. n= 3 mice per group. (F) Ki67, NeuN and EdU staining in the cerebellum of control and Sin3A CKO mice at P7. Nuclei were counterstained with DAPI. Mice were administrated EdU 48 hour before sacrifice. Arrowheads indicate cells positive for both NeuN and EdU, and arrows indicate cells positive for both Ki67 and EdU. The dashed lines delineate the boundaries of different layers within the cerebellar cortex. Scale bars indicate 250 μm. (G) Quantification of the percentage of EdU + /NeuN + double-positive cells relative to the total number of EdU + cells. Shown are mean ± SEM. Two-tailed t test: *** p ≤ 0.001. n= 3 mice per group. (H) Immunofluorescence analysis for Ki67 at P15 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bars indicate 250 μm. (I) Immunofluorescence staining for cleaved caspase-3 (CP3) and Ki67 at P0 and P7 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate CP3 + cells. Scale bars indicate 250 μm. (J) Quantification of the CP3 + cells in the EGL. Shown are mean ± SEM. Two-tailed t test: * p ≤ 0.05. n= 3 mice per group.

Techniques: Staining, Control, Two Tailed Test, Immunofluorescence

(A) Schematic representation of the strategy employed to isolate cells followed by high-throughput sequencing analysis. (B) Uniform Manifold Approximation and Projection (UMAP) visualization of cell populations from the P6 cerebellum of two Sin3A CKO and two control mice. UBC: unipolar brush cells, PC: purkinje cells, Oligo: oligodendrocytes, IN: GABAergic interneurons, Golgi: golgi cells, AC: astrocytes, BG: bergmann glial cells, (C) UMAP representation of the results from Milo differential abundance (DA) testing between Sin3A CKO and control cerebellum with nodes showing cell neighborhoods. Nodes with significant differential abundance are highlighted using a gradient scale of red or blue. (D) Beeswarm plot showing the log-fold change between Sin3A CKO and control cerebellum across groups of nearest-neighbor cells from various cell type clusters. Significant changes are highlighted using a gradient scale of red or blue. (E) Volcano plot illustrating the differential gene expression results between the GCP clusters of control and Sin3A CKO cerebellum. (F) Heatmap and average intensity plots of the Hdac1 ChIP-seq signals across 10 kb from the center of Sin3A peaks throughout the genome the genome from dissociated cerebellar cells of control mice. (G) Heatmap and average intensity plots of the H3K27ac ChIP-seq signals across 10 kb from the center of Sin3A peaks throughout the genome from isolated GCPs of control and Sin3A CKO mice. (H) Pie chart and Venn diagram illustrating the process of screening and identifying target genes of Sin3A. (I) Bar chart showing the results of the functional enrichment analysis for 96 candidate target genes.

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Schematic representation of the strategy employed to isolate cells followed by high-throughput sequencing analysis. (B) Uniform Manifold Approximation and Projection (UMAP) visualization of cell populations from the P6 cerebellum of two Sin3A CKO and two control mice. UBC: unipolar brush cells, PC: purkinje cells, Oligo: oligodendrocytes, IN: GABAergic interneurons, Golgi: golgi cells, AC: astrocytes, BG: bergmann glial cells, (C) UMAP representation of the results from Milo differential abundance (DA) testing between Sin3A CKO and control cerebellum with nodes showing cell neighborhoods. Nodes with significant differential abundance are highlighted using a gradient scale of red or blue. (D) Beeswarm plot showing the log-fold change between Sin3A CKO and control cerebellum across groups of nearest-neighbor cells from various cell type clusters. Significant changes are highlighted using a gradient scale of red or blue. (E) Volcano plot illustrating the differential gene expression results between the GCP clusters of control and Sin3A CKO cerebellum. (F) Heatmap and average intensity plots of the Hdac1 ChIP-seq signals across 10 kb from the center of Sin3A peaks throughout the genome the genome from dissociated cerebellar cells of control mice. (G) Heatmap and average intensity plots of the H3K27ac ChIP-seq signals across 10 kb from the center of Sin3A peaks throughout the genome from isolated GCPs of control and Sin3A CKO mice. (H) Pie chart and Venn diagram illustrating the process of screening and identifying target genes of Sin3A. (I) Bar chart showing the results of the functional enrichment analysis for 96 candidate target genes.

Techniques: Next-Generation Sequencing, Control, Gene Expression, ChIP-sequencing, Isolation, Functional Assay

(A) Immunofluorescence staining for Sox2, Atoh1-GFP, and NeuroD1 at E13.5 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate cells positive for Sox2 and Atoh1 in the EGL. Scale bar indicates 250 μm. (B) Immunofluorescence staining for Sox2, Atoh1-GFP, and NeuroD1 at E16.5 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Sox2 and Atoh1-GFP at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (D) Immunofluorescence staining for Sox2 and Atoh1-GFP at P7 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (E) Quantification of the Sox2 + ; Atoh1-GFP + cells relative to the Atoh1-GFP + population in the medial and anterior EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group. (F) Diagram illustrating the developmental model transitioning from multipotent ECPs to rapidly proliferating GCPs. (G) UMAP displaying the developmental trajectory of GC lineages from E12 to E16 wildtype mice, based on pseudotime analysis. (H) UMAP displaying the Sox2 and Atoh1 expression in the GC lineage cell of E12 to E16 wildtype mice. (I) Quantification of cells counts in the medial and anterior EGL of E16.5, P0 and P7 cerebellum. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, ** p ≤ 0.01. n= 3 or 4 mice per group.

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Immunofluorescence staining for Sox2, Atoh1-GFP, and NeuroD1 at E13.5 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate cells positive for Sox2 and Atoh1 in the EGL. Scale bar indicates 250 μm. (B) Immunofluorescence staining for Sox2, Atoh1-GFP, and NeuroD1 at E16.5 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Sox2 and Atoh1-GFP at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (D) Immunofluorescence staining for Sox2 and Atoh1-GFP at P7 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (E) Quantification of the Sox2 + ; Atoh1-GFP + cells relative to the Atoh1-GFP + population in the medial and anterior EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group. (F) Diagram illustrating the developmental model transitioning from multipotent ECPs to rapidly proliferating GCPs. (G) UMAP displaying the developmental trajectory of GC lineages from E12 to E16 wildtype mice, based on pseudotime analysis. (H) UMAP displaying the Sox2 and Atoh1 expression in the GC lineage cell of E12 to E16 wildtype mice. (I) Quantification of cells counts in the medial and anterior EGL of E16.5, P0 and P7 cerebellum. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, ** p ≤ 0.01. n= 3 or 4 mice per group.

Techniques: Immunofluorescence, Staining, Control, Two Tailed Test, Expressing

(A) Bulk RNA-seq tracks (reads per million values) showing the expression level of Atoh1 in the GCPs of control and Sin3A CKO mice. ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, and H3K27ac occupation at the Sox2 locus in the dissociated cerebellar cells or isolated GCPs. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments; absence of genotype annotation indicates a wild-type or Atoh1 M1GFP/+ animal. (B) Violin plots showing the expression of Sox2 in the GCP population of Sin3A CKO and control cerebellum. (C) Splitted UMAP displaying the Sox2 expression in the sub-clusters of GCPs between Sin3A CKO and control. (D) Sox2, Atoh1-GFP, and EdU staining in the cerebellum of control and Sin3A CKO mice at P0. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate cells positive for both Sox2 and EdU, and arrows indicate cells positive for both Atoh1-GFP and EdU. Scale bar indicates 250 μm. (E) Quantitative analysis showing the percentage of EdU + cells relative to the Sox2 + ; Atoh1-GFP + or Atoh1-GFP + population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, *** p ≤ 0.001. n= 3 mice per group. (F) Schematic representation of the strategy employed to overexpress Sox2 using electroporation. (G) NeuroD1, tdTomato and Sox2 staining in the cerebellum of electroporated mice harvested at P5. Arrowheads indicate tdTomato + cells in the EGL. Scale bar indicates 20 μm. (H) Quantitative analysis showing the percentage of EdU + cells relative to the tdTomato + ; Ki67 + population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05. n= 3 mice per group. (I) tdTomato and EdU staining in the cerebellum of electroporated mice at P5. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate tdTomato + cells in the EGL. Scale bar indicates 20 μm.

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Bulk RNA-seq tracks (reads per million values) showing the expression level of Atoh1 in the GCPs of control and Sin3A CKO mice. ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, and H3K27ac occupation at the Sox2 locus in the dissociated cerebellar cells or isolated GCPs. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments; absence of genotype annotation indicates a wild-type or Atoh1 M1GFP/+ animal. (B) Violin plots showing the expression of Sox2 in the GCP population of Sin3A CKO and control cerebellum. (C) Splitted UMAP displaying the Sox2 expression in the sub-clusters of GCPs between Sin3A CKO and control. (D) Sox2, Atoh1-GFP, and EdU staining in the cerebellum of control and Sin3A CKO mice at P0. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate cells positive for both Sox2 and EdU, and arrows indicate cells positive for both Atoh1-GFP and EdU. Scale bar indicates 250 μm. (E) Quantitative analysis showing the percentage of EdU + cells relative to the Sox2 + ; Atoh1-GFP + or Atoh1-GFP + population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, *** p ≤ 0.001. n= 3 mice per group. (F) Schematic representation of the strategy employed to overexpress Sox2 using electroporation. (G) NeuroD1, tdTomato and Sox2 staining in the cerebellum of electroporated mice harvested at P5. Arrowheads indicate tdTomato + cells in the EGL. Scale bar indicates 20 μm. (H) Quantitative analysis showing the percentage of EdU + cells relative to the tdTomato + ; Ki67 + population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05. n= 3 mice per group. (I) tdTomato and EdU staining in the cerebellum of electroporated mice at P5. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate tdTomato + cells in the EGL. Scale bar indicates 20 μm.

Techniques: RNA Sequencing, Expressing, Control, ChIP-sequencing, Isolation, Staining, Two Tailed Test, Electroporation

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Bulk RNA-seq tracks (reads per million values) showing the expression level of Atoh1 in the GCPs of control and Sin3A CKO mice. ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, and H3K27ac occupation at Atoh1 locus in the dissociated cerebellar cells or isolated GCPs. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments; absence of genotype annotation indicates a wild-type or Atoh1 M1GFP/+ animal. Enhancers A and B are two critical regulatory elements located downstream of the Atoh1 coding region, identified through reported gene assay and cross-species conservation sequence analysis . (B) Immunofluorescence staining for Atoh1-Cre at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Atoh1-GFP and Ki67 at P7 and P15 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 250 μm. (D) Boxplot showing the quantification of the relative fluorescence intensity of GFP or Cre in EGL cells of control and Sin3A CKO mice. Two-tailed t test: **** p ≤ 0.0001. n= 200 cells per group. (E) Flow cytometry analysis of GCPs isolated from Atoh1-Cre; Atoh1 M1GFP/+ and Sin3A CKO ; Atoh1 M1GFP/+ mice. Cells were dissociated from the anterior regions of the cerebellum in P7 mice and subsequently sorted based on endogenous GFP expression. (F) Single-molecule fluorescence in situ hybridization (smFISH) analysis showing the transcription levels of Atoh1 in EGL of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (G) Quantification of the count of Atoh1 transcripts per 100 square micrometers. Shown are mean ± SEM. Two-tailed t test: ** p≤ 0.01. n= 3 mice per group.

Techniques: RNA Sequencing, Expressing, Control, ChIP-sequencing, Isolation, Gene Assay, Sequencing, Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Flow Cytometry, In Situ Hybridization

(A) Venn diagram illustrating the identification of genomic loci directly regulated by the Sin3A/Hdac1 complex. (B) Heatmap and average intensity plots illustrating the H3K27ac ChIP-seq signal profiles surrounding 3439 genomic loci directly regulated by the Sin3A/Hdac1 complex in isolated GCP from both control and Sin3A CKO mice. (C) Selected motifs exhibit significant enrichment at genomic loci that are directly regulated by the Sin3A/Hdac1 complex. (D) Immunofluorescence staining for Atoh1-GFP and NeuroD1 at P6 in the cerebellum of control and Sin3A CKO mice. Scale bar indicates 250 μm. (E) Venn diagram illustrating the overlaps of Sin3A, Hdac1 and NeuroD1 binding sites in the genome of wild type cerebellum. (F) ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, NeuroD1, and H3K27ac occupation at Atoh1 locus in the GCP of control, Sin3A CKO , and NeuroD1 LacZ/LacZ mice. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments, absence of genotype annotation indicates a wild-type animal. (G) Immunoblots for NeuroD1, Sin3A and Hdac1 binding in P7 cerebellum of wild-type mice. Lane 1-3: cell lysate of P7 cerebellum. Lane 4: Co-immunoprecipitation of rabbit IgG in cell lysate of P7 cerebellum. Lane 5: Co-immunoprecipitation of NeuroD1 in cell lysate of P7 cerebellum. (H) Immunofluorescence staining for Atoh1-GFP, NeuN, and Ki67 at P6 in the cerebellum of control and NeuroD1 LacZ/LacZ mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (I) Boxplot showing the quantification of the relative fluorescence intensity of GFP in EGL cells of control and NeuroD1 lacZ/lacZ mice. Two-tailed t test: **** p ≤ 0.0001. n= 200 cells per group. (J) smFISH analysis showing the transcription levels of Atoh1 in the EGL of wild type and NeuroD1 lacZ/lacZ mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (K) Quantification of the count of Atoh1 transcripts per 100 square micrometers in the EGL of wild type and NeuroD1 lacZ/lacZ mice. Shown are mean ± SEM. Two-tailed t test: ** p≤ 0.01. n= 3 mice per group. (L) Heatmap and average intensity plots of the H3K27ac ChIP-seq signals across 10 Kb from the center of Sin3A/Hdac1/NeuroD1 binding sites throughout the genome from isolated GCPs of control and NeuroD1 lacZ/lacZ mice.

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Venn diagram illustrating the identification of genomic loci directly regulated by the Sin3A/Hdac1 complex. (B) Heatmap and average intensity plots illustrating the H3K27ac ChIP-seq signal profiles surrounding 3439 genomic loci directly regulated by the Sin3A/Hdac1 complex in isolated GCP from both control and Sin3A CKO mice. (C) Selected motifs exhibit significant enrichment at genomic loci that are directly regulated by the Sin3A/Hdac1 complex. (D) Immunofluorescence staining for Atoh1-GFP and NeuroD1 at P6 in the cerebellum of control and Sin3A CKO mice. Scale bar indicates 250 μm. (E) Venn diagram illustrating the overlaps of Sin3A, Hdac1 and NeuroD1 binding sites in the genome of wild type cerebellum. (F) ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, NeuroD1, and H3K27ac occupation at Atoh1 locus in the GCP of control, Sin3A CKO , and NeuroD1 LacZ/LacZ mice. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments, absence of genotype annotation indicates a wild-type animal. (G) Immunoblots for NeuroD1, Sin3A and Hdac1 binding in P7 cerebellum of wild-type mice. Lane 1-3: cell lysate of P7 cerebellum. Lane 4: Co-immunoprecipitation of rabbit IgG in cell lysate of P7 cerebellum. Lane 5: Co-immunoprecipitation of NeuroD1 in cell lysate of P7 cerebellum. (H) Immunofluorescence staining for Atoh1-GFP, NeuN, and Ki67 at P6 in the cerebellum of control and NeuroD1 LacZ/LacZ mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (I) Boxplot showing the quantification of the relative fluorescence intensity of GFP in EGL cells of control and NeuroD1 lacZ/lacZ mice. Two-tailed t test: **** p ≤ 0.0001. n= 200 cells per group. (J) smFISH analysis showing the transcription levels of Atoh1 in the EGL of wild type and NeuroD1 lacZ/lacZ mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (K) Quantification of the count of Atoh1 transcripts per 100 square micrometers in the EGL of wild type and NeuroD1 lacZ/lacZ mice. Shown are mean ± SEM. Two-tailed t test: ** p≤ 0.01. n= 3 mice per group. (L) Heatmap and average intensity plots of the H3K27ac ChIP-seq signals across 10 Kb from the center of Sin3A/Hdac1/NeuroD1 binding sites throughout the genome from isolated GCPs of control and NeuroD1 lacZ/lacZ mice.

Techniques: ChIP-sequencing, Isolation, Control, Immunofluorescence, Staining, Binding Assay, Western Blot, Immunoprecipitation, Fluorescence, Two Tailed Test

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