Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: MSCs were exposed to increasing LPS concentrations, thereafter washed and co‐cultured with PMA‐activated neutrophils. Incubation of neutrophils with PMA alone served as a positive control and DMSO‐treated neutrophils as a negative control. Scale bars: 50 μm. Quantification of NET‐bound elastase indicative of NET formation. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. Representative microphotographs of murine wound sections immunostained for Ly6G (neutrophils, green) and DNA‐histone (displayed as expulsed streaks in red indicative of NETs). Wounds injected with LPS‐primed MSCs (upper row, outer right panel) depict enhanced NET formation (magnified inset) and increased expression of activation markers like neutrophil elastase (NE, lower panel, outer left panel) compared to wounds injected with non‐primed MSC (middle panels) or PBS‐injected wounds (outer left panels). Murine skin wounds treated with PBS injections served as control. Scale bar: 10 μm (upper row) and 50 μm (lower row). To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Cell Culture, Incubation, Positive Control, Negative Control, Injection, Expressing, Activation Assay, Control, Comparison

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Graph shows quantification of ROS by measuring fluorescence intensity of DHR123 dye. The MSCs were primed with increasing concentrations of 10 ng/ml, 100 ng/ml, and 1,000 ng/ml LPS followed by co‐culture with neutrophils. The MSCs were then incubated with ROS‐specific dye DHR123, and the fluorescence was read after 60 min of incubation at 488/520 nm with spectrophotometer. Incubation of neutrophils with PMA alone served as a positive control and DMSO‐treated neutrophils as a negative control. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Fluorescence, Co-Culture Assay, Incubation, Spectrophotometry, Positive Control, Negative Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: A Hierarchical clustering analysis of RNAseq expression profile from MSCs stimulated with LPS for 6 and 24 h. The heat‐map shows the gene expression profile of non‐primed MSCs and MSCs which were LPS primed for 6 and 24 h. Red color represents up‐regulation, while blue color depicts down‐regulation in gene expression; each data point represents FPKM in log 2 value. B, C Pathway analysis depicts that cytokine–cytokine receptor interactions, genes encoding secreted soluble factors, an ensemble of genes encoding extracellular matrix, chemokine receptors which bind distinct chemokines, GPCR ligand binding, cytokine and inflammatory response, chemokine signaling pathway and IL‐5 signaling are the most dominant pathways as assessed by transcriptional changes when non‐primed and LPS‐primed MSCs were compared at 6 and 24 h after LPS priming. D Venn diagram analysis displays up‐regulated and down‐regulated gene numbers indicated in blue and pink colors, respectively. Purple color indicates the number of shared genes of the up‐regulated and down‐regulated genes at 6 h and 24 h after LPS stimulation. This implies rapid changes of expression of the same genes. E–G Validation of the selected genes uncovered from global RNAseq analyses by qRT–PCR analyses displays up‐regulation of CXCL‐6, IL‐8, and IL‐1β after LPS treatment. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. H, I ELISA results depict an up‐regulated expression of CXCL‐6 after 24 h of LPS treatment, while a significantly increased IL‐8 expression after 6 and 24 h of LPS treatment. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. J Western blot analysis showed up‐regulation of IL‐1β expression upon LPS priming of LPS‐primed MSCs. Actin served as control. Densitometry graph shows significant increase after 6 h of LPS stimulation compared to non‐primed MSCs. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, n = 3 biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Expressing, Gene Expression, Ligand Binding Assay, Biomarker Discovery, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Human MSCs were treated with LPS (100 ng/ml) for 24 h. The mRNA expression of MyD88 and IL‐6 was assessed by qRT–PCR analysis at 24 h. MyD88 (upper left panel) and IL‐6 gene expression (upper right panel) revealed an increase in MyD88 and IL‐6 in LPS‐primed MSCs. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. The TLR4 signaling pathway is depicted. Upon LPS binding, conformational changes in the TLR4 receptor led to recruitment of intracellular TLR domains harboring adaptor proteins. A bifurcation in MyD88‐dependent signaling converges to NF‐κB and pro‐inflammatory cytokine release, among them IL‐6, and the TRIF signaling pathway relaying its signals to downstream effectors like the transcription factor IRF which transactivates type 1 interferons. The right panel depicts representative Western blot analyses with a time‐dependent regulation of TLR4 and components of the TLR4 signaling pathway after exposure of MSCs with LPS. p65, a component of the heterodimeric NF‐κB transcription factor, and the IRF‐1 transcription factor are swiftly induced at 0.5 h, but down‐regulated to basal levels at 6 h after LPS challenge of MSCs. The target genes of NF‐κB are induced at 0.5 h, and this induction is differentially maintained. The expression of indicated proteins was analyzed in the cell lysates from LPS (100 ng/ml)‐ or PBS‐treated MSCs, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Binding Assay, Western Blot

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS‐primed MSCs into wounds increased the expression of CXCL6, IL‐8, and IL‐1β. Graphs display numbers of double‐positive cells for h‐β2M + CXCL6, h‐β2M + IL‐8, and h‐β2M + IL‐1β, respectively. MSC‐injected wounds served as controls. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50 μm. Results show that LPS‐primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL‐8 in mice and is known as neutrophil chemoattractant. Similar results were found for IL‐1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP‐2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double‐positive cells for Ly6G MIP/KC, IL‐1β, and LIX, respectively. PBS and non‐primed MSC‐injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Scale bars: 50 μm.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Injection, Expressing, Comparison, Functional Assay, Sequencing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: MSCs were either treated with a scrambled control siRNA (Scr. siRNA) or with four different siRNAs targeting the TLR4 receptor (siRNA smart pool). Forty‐eight hours after transfection, TLR4‐silenced and non‐silenced MSCs were analyzed for RNA expression by qPCR. A representative experiment is depicted. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. Seventy‐two hours after transfection, TLR4‐silenced and non‐silenced MSCs were treated with 100 ng/ml LPS for 16 h and subjected to Western blot analysis for protein expression. Densitometric assessment shows the percentage of the change in TLR4 expression in TLR4‐silenced and non‐silenced MSC. A representative experiment is depicted. Statistical analysis was performed using unpaired t ‐test, and values are represented as mean ± SEM, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Control, Transfection, RNA Expression, Western Blot, Expressing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative microphotographs of TLR4‐silenced and non‐silenced MSCs primed with LPS and co‐cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA‐histone, red). Of note, TLR4‐silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non‐silenced, LPS‐primed MSCs co‐cultured with PMA‐activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co‐culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50 μm. Quantitative assessment of NET‐bound elastase employing a specific ELISA in identical experimental setting as described in Fig . Similarly, to immunostaining, a significant reduction of NET‐bound elastase was observed in TLR4‐silenced LPS‐primed MSCs when co‐cultured with activated neutrophils as opposed to high NET‐bound elastase in non‐silenced LPS‐primed MSCs co‐cultured with activated neutrophils. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates. High‐resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS‐primed MSC co‐cultured with PMA‐activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non‐primed MSCs co‐cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4‐silenced LPS‐primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4‐silenced and non‐primed MSCs display reduced NETs in co‐cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA‐activated neutrophils alone served as a positive control with highly enhanced NETs (upper row, outer right panel) and DMSO‐treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil‐derived granules. Scale bars: 0.1 μm

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Cell Culture, Immunostaining, Negative Control, Positive Control, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Microscopy, Activation Assay, Derivative Assay

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative clinical pictures of murine wounds at 0, 3, 5, 7, and 10 days after wounding. Enhanced wound healing in the LPS‐primed MSCs group as opposed to all other groups. Statistical analysis of 20 wound areas per group at the indicated time points, expressed as percentage of the initial wound size (day 0), for PBS control, non‐primed MSCs, LPS‐primed MSCs, non‐primed scrambled siRNA‐treated (Scr) MSCs, LPS‐primed Scr MSCs, and LPS‐primed TLR4‐silenced MSCs. Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments. Statistical analysis was performed using one‐way ANOVA. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G + neutrophils (green) and F4/80 + macrophages (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 1 wounds injected with PBS (control), non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Double‐stained cells indicate phagocytic engulfment of neutrophils by macrophages. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bar: 100 μm. Quantification of Ly6G and F4/80 double‐positive cells on sections of differently injected day 1 wounds. Wounds were injected as described in (A). Double‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Quantification of Ly6G + neutrophils and F4/80 + macrophages on sections of differently injected day 1 wounds. Wounds were injected as described in (A). Single‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double‐stained for TGFβ‐1 (red) and for F4/80 + macrophages (green). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 5 wounds injected with PBS (control), non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Scale bar: 50 μm. Representative photomicrographs of sections of day 5 wounds immunostained for CD31 (indicative of endothelial cells and newly formed vessels) and for α‐SMA (indicative of myofibroblasts differentiation) after injection of LPS‐primed MSCs, non‐primed MSCs, LPS‐primed TLR4‐silenced MSCs or PBS (middle and lower panel). To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bars: 50 μm. Double‐positive macrophages stained for TGFβ‐1 and F4/80 were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Quantitative analysis of CD31‐positive endothelial cells in sections of wounds injected with PBS, non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced LPS. Cell counting was performed on immunostained wound sections; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates. Western blot analysis of lysates from day 5 wounds (left panel) and the corresponding densitometric analysis (right panel) depict enhanced α‐SMA protein expression in wounds injected with LPS‐primed MSCs as opposed to the respective control groups. Actin served as loading control. Statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, three biological replicates. Source data are available online for this figure.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Confocal Microscopy, Injection, Staining, Double Staining, Control, Comparison, Cell Counting, Western Blot, Expressing

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Representative photomicrographs of sections from differently injected day 3 wounds stained for Ly6G + neutrophils (green) and caspase 3 (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 3 wounds injected with PBS, non‐primed MSCs, LPS‐primed MSCs, and LPS‐primed TLR4‐silenced MSCs. Double‐stained cells indicate apoptotic neutrophils. To facilitate comparison, areas inside the rectangles are shown at 5× magnification in the insets. Scale bars: 50 μm. Quantification of Ly6G and caspase double‐positive cells on sections of differently injected day 3 wounds. A significant increase in double‐positive cells (Ly6G and caspase 3) in sections of wounds injected with LPS‐primed MSCs as opposed to wounds injected with LPS‐primed TLR4‐silenced MSCs and PBS control. Double‐positive cells were counted; statistical analysis was performed using one‐way ANOVA, and values are represented as mean ± SEM, six biological replicates.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Injection, Staining, Double Staining, Comparison, Control

Journal: EMBO Reports

Article Title: TLR4‐dependent shaping of the wound site by MSCs accelerates wound healing

doi: 10.15252/embr.201948777

Figure Lengend Snippet: Graphical summary, depicting the molecules involved in sensing, signaling, and raising an adaptive response in MSCs. MSCs sense bacterial intruders as modeled by the key molecule LPS, a widely distributed PAMP and wall component of Gram‐negative bacteria. LPS is sensed via the TLR4 receptor, and the signal is relayed via MyD88 to downstream effectors which, in consequence, shape the adaptive MSC response. This response is enforced by fundamental transcriptomic reprogramming which is responsible for the release of factors critical for neutrophil and macrophage recruitment to the wound site. The adaptive response of LPS‐primed MSCs depicts a bifurcation with the activation of neutrophils with enhanced microbicidal NET formation and ROS release to directly counteract invading bacteria. In addition, in a second line of tissue protection and repair, apoptotic neutrophils are phagocytosed by macrophages. Neutrophil engulfment constitutes a strong signal for macrophages to release TGFβ‐1 which subsequently enhances differentiation of myofibroblasts and accelerates wound contraction and thus wound closure. Acceleration of wound closure together with enhanced NET formation and ROS release effectively counteracts microbial invasion. These data may stimulate new avenues to refine MSC‐based therapies for difficult‐to‐heal wounds and/or infected wounds.

Article Snippet: Human adipose‐derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC) (LGC Standards GmbH, Wesel Germany).

Techniques: Bacteria, Activation Assay, Infection

Journal: Nucleic Acids Research

Article Title: Ecd promotes U5 snRNP maturation and Prp8 stability

doi: 10.1093/nar/gkaa1274

Figure Lengend Snippet: Ecd Δ34 mutant protein binds R2TP complex via PIH1D1 but is non-functional in vivo . ( A ) Schematic representation of wild-type and mutant Ecd proteins and ecd alleles used in this study. The blue boxes indicate the position of wild-type (dark blue) or mutated (light blue) DSDD/DEDD motifs. Alignment of the C-terminal part of the Drosophila melanogaster ( D.m ., Q9W032) Ecd and human ( H.s. , O95905) ECD proteins was generated using Clustal W (A’). Asterisks indicate the positions of the premature stop codon in ecd l( 3 )23 allele and the conserved proline 656, which substitution to serine generates conditional ecd 1 allele. Black and red rectangles outline the DSDD and DEDD motifs, respectively (A’). ( B , C ) Representative western blots and quantifications show that Myc::Ecd TripleA (B) but not Myc::Ecd Δ34 (C) co-precipitates significantly less Flag::PIH1D1 protein from Drosophila S2 cell lysates relative to Myc::Ecd wt (B, C). Myc-tagged proteins served as baits. GFP was used as a transfection and loading control. Data represent means ± SD, n = 3. Unpaired two-tailed Student's t -test was used to determine the significance, *** P < 0.001, n.s. = non-significant. ( D–M ) Representative confocal micrographs of mosaic third instar EADs and brightfield and fluorescent images of adult eyes, where homozygous GFP-labelled clones of the indicated genotypes were generated using the eyFLP MARCM technique. In contrast to abundant, sizable control clones (D, E), ecd Δ homozygous mutant clones are very rare, presented as individual GFP-positive cells within the differentiated part of the eye primordium (F) and adult retina (G). Overexpression of Ecd wt (H, I) and Ecd TripleA (J, K) but not Ecd Δ34 mutant protein (L, M) is sufficient to restore clonal number and size to control levels (D, E). Confocal micrographs are projections of multiple sections, showing EADs 7 days AEL. Nuclei were counterstained with DAPI. Scale bars: 100 μm (D, F, H, J, L). ( N ) Quantification of clonal to total EAD volume ratios from confocal micrographs of mosaic EADs of the indicated genotypes. Data represent means ± SD, n = 7–16. Ordinary one-way ANOVA with Tukey's multiple comparisons test was used to determine significance, **** P < 0.0001, n.s. = non-significant. See also .

Article Snippet: Drosophila melanogaster coding DNA sequences of PIH1D1 (CG5792), Pontin (CG4003), SmD3 (CG8427), SmB (CG5352), SmG (CG9742), SmF (CG16792) and Ecd (CG5714) were amplified from respective cDNAs with Phusion HS II polymerase (ThermoFisher Scientific Cat# F549L) and cloned into the pENTR4 dual selection vector (ThermoFisher Scientific Cat# A10465).

Techniques: Mutagenesis, Functional Assay, In Vivo, Generated, Western Blot, Transfection, Control, Two Tailed Test, Clone Assay, Over Expression

Journal: Cell

Article Title: Atlas of Subcellular RNA Localization Revealed by APEX-seq

doi: 10.1016/j.cell.2019.05.027

Figure Lengend Snippet: (A) APEX2-mediated proximity biotinylation of endogenous RNAs. APEX2 peroxidase is genetically targeted to the cellular region of interest. Addition of biotin-phenol (red B = biotin) and H2O2 to live cells for 1 minute results in biotinylation of endogenous proteins and RNA within a few nanometers of APEX2. Biotinylated RNAs are separated using streptavidin-coated beads, polyA-selected, and analyzed by RNA-seq.

Article Snippet: To enrich biotinylated RNAs we used Pierce streptavidin magnetic beads (Thermo Fischer), using 10 μl beads per 25 μg of RNA.

Techniques: RNA Sequencing Assay

Journal: Cell

Article Title: Atlas of Subcellular RNA Localization Revealed by APEX-seq

doi: 10.1016/j.cell.2019.05.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To enrich biotinylated RNAs we used Pierce streptavidin magnetic beads (Thermo Fischer), using 10 μl beads per 25 μg of RNA.

Techniques: Recombinant, Magnetic Beads, Sample Prep, RNA Sequencing Assay, Fractionation, Blocking Assay, Plasmid Preparation, Sequencing, Software

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 1. The FoxP3 Reporter LV Shows Expression Selective for the Treg Cell Lineage (A) Endogenous human FOXP3 gene shows location of regulatory regions (promoter, CNS1, CNS2, CNS3, and 30 UTR) included in vector. Vector maps show design of CNS123p-mStrawberry and CNS123p-FoxP3-mStrawberry constructs within the pCCL vector backbone. (B) Analysis of human hematopoietic cell lines transduced with CNS123p-mStrawberry. Histograms show endogenous FoxP3 status of each cell line (left) and mStrawberry expression (right) in each cell line transduced with CNS123p-mStrawberry. Plots show mStrawberry expression in human hematopoietic cell lines over a range of vector copy numbers (n = 12 per cell line). (C) Activated human CD4 cells transduced with different doses of CNS123p-mStrawberry. Histograms show mStrawberry expression in viable CD4+ cells analyzed 4 days after activation. (see also Figure S2B). (D) Experimental design to evaluate in vivo lineage-specific expression of CNS123p-mStrawberry. Lin HSPCs were isolated from CD45.2 FoxP3-prom-GFP mice and transduced with CNS123p-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated congenic CD45.1 recipients. CD45.2 donor cells within each hematopoietic lineage were analyzed at 20 weeks post-transplant for mStrawberry reporter LV expression (see also Figure S2A). (E) Histograms depict mStrawberry reporter expression in each hematopoietic lineage in bone marrow, thymus, and spleen of engrafted mice. Each individual histogram line represents mStrawberry expression for an individual mouse (n = 9 mice; see also Figure S2C). (F) y axis represents the percentage of mStrawberry+ cells within each hematopoietic lineage in the BM, spleen, and thymus (n = 9 mice). Data in (E) represent mean ± SD.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Plasmid Preparation, Construct, Transduction, Activation Assay, In Vivo, Isolation, Irradiation

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 2. Lineage-Specific FoxP3 Expression Restores Treg Cell Development from Scurfy (FoxP3 Mutant) HSCs (A) Transplant setup to evaluate Treg cell development. Scurfy (FoxP3mut) mice were rescued with WT CD45.1 splenocytes at birth to allow survival into adulthood to serve as bone marrow donors. Lin HSPCs were isolated from rescued scurfy (FoxP3mut) or wild-type (FoxP3-prom-GFP) donor mice and transduced with CNS123p-mStrawberry or CNS123p-FoxP3-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated WT CD45.1 congenic recipients. After 12 weeks, donor cells from each transplant cohort were evaluated for thymic and splenic reconstitution of Treg cells. Treg cell populations from each group were identified as CD4+mStrawberry+ cells (uncorrected scurfy Treg cells [Sf-Treg cells]; corrected scurfy Treg cells [cSf-Treg cells]) or CD4+GFP+ cells (wild- type Treg cells [WT-Treg cells]). (B) Lineage distribution of total donor thymocytes in mice reconstituted with Sf, cSf, or WT BM lin cells (n = 3–5 mice/arm). (C) Thymic Treg cell reconstitution. FACS plots show donor CD45.2+CD4SP cells in the thymus of transplant recipients. Gates delineate thymic Sf-Treg cells, cSf-Treg cells, and WT-Treg cells. Bottom panel shows expression of the Treg cell surface markers CD25, GITR, and CTLA4 within each putative Treg cell population (surface marker expression for Sf-Treg cells and cSf-Treg cells is shown for mStrawberryhigh cells or cells expressing the top 50% of mStrawberry expression). Histograms depict one representative experiment of 3 (see also Figure S2D). (D) FACS sort for splenic Treg and Tconv cells. Splenic Treg cell populations (mStrawberryhigh or GFP+ gates) were FACS sorted for a Treg cell suppression assay. Tconv cell populations (mStrawberry or GFP gates) were sorted for an iTreg cell induction assay. (E) In vitro Treg cell suppression assay. Sorted Treg cells (shown in D) were co-cultured with responder T cells (Tresp cells) (congenic WT CD4+ cells labeled with a fluorescent proliferation dye) at a 1:1 ratio in the presence of bead-bound CD3 and CD28 antibodies. Histograms depict Tresp cell proliferation in one of three representative experiments. Bar graph shows proliferation index for each Treg cell culture condition (n = 6–9 Tresp cell cultures per arm from 3 different Treg cell sources per arm; data normalized to internal ‘‘no Treg cell’’ control for each experiment). (F) Sorted splenic Tconv cells (shown in D) were activated with CD3 and CD28 antibodies in the presence of 20 ng/mL interleukin-2 (IL-2) and 20 ng/mL TGF-b for 4 days to induce iTreg cells. FACS plots show mStrawberry or GFP expression from each group (n = 3 mice/group). Data in (B), (C), (E), and (F) are presented as mean ± SD. Data in (B) were analyzed by Kruskal-Wallis test for each lineage. Data in (E) are analyzed by Kruskal- Wallis test for overall comparison for all groups and Mann-Whitney U test for pairwise comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Mutagenesis, Isolation, Transduction, Irradiation, Marker, Suppression Assay, In Vitro, Cell Culture, Labeling, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 3. The Lineage-Specific FoxP3 cDNA Vector Generates Functional Treg Cells Capable of Rescuing the Scurfy Mouse (A) Assay for in vivo Treg cell function: three groups of CD4 cells containing putative Treg cells were generated by congenic bone marrow transplants: uncorrected scurfy CD4 (Sf-CD4; no. 1); corrected scurfy CD4 (cSf-CD4; no. 2); and wild-type CD4 (WT-CD4; no. 3). To obtain Sf-CD4 and cSf-CD4, CD45.2 Lin HSPCs were isolated from scurfy donors (rescued at birth by CD45.1 splenocytes) and transduced with either CNS123p-mStrawberry (Sf-CD4; no. 1) or CNS123p-FoxP3- mStrawberry (cSf-CD4; no. 2). To obtain WT-CD4, CD45.2 Lin HSPCs were isolated from FoxP3-prom-GFP donors and transduced with CNS123p- mStrawberry (no. 3). Transduced cells were transplanted into lethally irradiated congenic (CD45.1) recipients. 8 weeks post-transplant, donor CD45.2+CD4+ cells were purified with magnetic beads from the spleens of transplant recipients and injected intraperitoneally into scurfy neonates. Scurfy neonates and age-matched WT receiving PBS injection were also analyzed as control conditions no. 4 and no. 5. The autoimmune phenotype of all groups was evaluated at 21 days of life (see also Figure S3). (B) Photographs of scurfy mice or WT controls at 21 days. White arrows highlight ear skin phenotype in mice from each group. Ear skin inflammation (char- acterized by small, thickened, scaly ears) is seen in untreated scurfy mice (Sf + PBS) and scurfy mice receiving uncorrected scurfy CD4 cells (‘‘Sf + Sf-CD4’’). Normal ear skin without inflammation is seen in WT controls (‘‘WT + PBS’’) and scurfy mice receiving wild-type or corrected scurfy CD4 cells (‘‘Sf + WT-CD4’’ and ‘‘Sf + cSf-CD4’’). (C) Spleen-to-body-weight ratio for rescued scurfy mice or WT littermate controls. (D) Activated (CD44+CD62L) CD4 T cells (expressed as a percentage of total CD4 splenocytes) in the spleens of rescued scurfy mice or WT littermate controls. (E) Serum cytokine levels in rescued scurfy mice or WT littermate controls. Data in (C)–(E) are presented as mean ± SD. Data on (C)–(E) represent n = 3 independent experiments pooled for analysis with a total of 3–6 mice/arm. Data in (C)–(E) were analyzed by Kruskal-Wallis test for overall comparison for all groups, and Mann-Whitney U test was performed for pairwise comparisons. *p < 0.05; **p < 0.01.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Functional Assay, In Vivo, Cell Function Assay, Generated, Isolation, Transduction, Irradiation, Magnetic Beads, Injection, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 4. The FoxP3 Reporter Vector CNS123p-mStrawberry Shows Treg Cell Lineage-Selective Expression in a Humanized Mouse Model (A) Experimental setup for humanized mouse models. Cord blood CD34+ HSPCs were transduced with CNS123p-mStrawberry and transplanted into neonatal NSG mice (B, C, E, and F) or NSG-SGM3 mice (D). 12–16 weeks post-transplant, engrafted hCD45+ cells were analyzed for mStrawberry expression. (B) mStrawberry reporter expression in each hematopoietic lineage. Each overlaid histogram represents mStrawberry expression in an individual mouse (n = 10–14 mice humanized with 2 different cord blood CD34+ donors; see also Figures S4A–S4C). (C) Percentage of mStrawberry+ cells in each lineage shown in (B). (D) Co-expression of FoxP3 and mStrawberry in humanized mice. Splenic human CD4+ cells were FACS sorted into mStrawberry+ and mStrawberry pop- ulations followed by intracellular staining for FoxP3 expression. Left panel shows sorting of human CD4+ cells by mStrawberry expression, and right panel shows FoxP3 expression in sorted populations. Results are representative of 2 independent experiments. (E) CNS2 methylation analysis of T cell populations from humanized mice. Figure shows the locations of CNS2 within the endogenous FOXP3 gene and CNS2 within the viral genome. Red arrows indicate differential primer binding sites for amplification of endogenous or viral CNS2. FACS plot shows sorting gates used to define Treg cell (CD4+CD25+) and Tconv cell (CD4+CD25) populations in CD4-enriched cells isolated from the pooled spleens of 3–5 humanized mice. Heatmap represents the percentage of methylated reads detected at each of the 9 CpG sites within endogenous and viral CNS2. Results are representative of 2 independent experiments using pooled NSG cohorts humanized from 2 different CB CD34+ donors (see also Figures S4D–S4F). (F) Experimental setup for NSG competitive repopulation assay. ‘‘Test’’ CB CD34+ cells were transduced with either CNS123p-mStrawberry or CNS123p-FoxP3- mStrawberry, and ‘‘competitor’’ CD34+ cells were transduced with a UBC-mCitrine vector. Test and competitor cells were co-transplanted at a 1:1 ratio into NSG neonates, and the percentage of competitor (mCitrine+) CD45+ cells engrafted in the BM at 12 weeks was determined for each group (n = 6 mice per group; humanized from 2 different CB CD34+ donors). Data in (F) represent mean ± SD. Data in (F) were analyzed by Mann-Whitney U test.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Expressing, Transduction, Staining, Methylation, Binding Assay, Isolation, MANN-WHITNEY

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm. (B) Functional annotation of AMIGO2-interacting proteins detected by GFP pull-down followed by MS in SKmel147 cells stably expressing AMIGO2-GFP (see Table S4). (C) PTK7 and GFP immunoblots following GFP pull-down from 501MEL cells stably expressing AMIGO2-GFP. (D) Full-length PTK7 (FL-PTK7), C-terminal fragments CTF1- and CTF2-PTK7, and FOXM1 immunoblots of 501MEL cells 72 hr post-infection with shSCR or shPTK7 (shP7 #1 and #2). Actin was used as a loading control. (E) Relative growth curves of 501MEL (left) and SKmel147 (right) cells stably transduced with shSCR or shPTK7 (shP7 #1 and #2). Values are normalized to seeding control (n = 3). (F) Percent Annexin V-positive cells 6 days post-transduction for same cells as in (E). (G) FL-PTK7, CTF-PTK7, and FOXM1 immunoblots of 501MEL cells 48 hr post-transduction with shSCR or shAMIGO2 (shA2 #1 and #2). Actin was used as a loading control. (H) FL-PTK7, CTF-PTK7, FOXM1, and AMIGO2 immunoblots of 501MEL cells untreated or treated with JQ1 (JQ1[+]) for 72 hr. Tubulin was used as a loading control. (I) CTF2-PTK7 immunoblot of nuclear lysates from same cell as in (G) (left). Lamin B1 was used as loading control. Signal quantification (right), normalized to Lamin B1, relative to shSCR (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S3 and S4.

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Functional Assay, Western Blot, Infection, Control, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, DNA Library Preparation, Blocking Assay, Extraction, TA Cloning, Sequencing, RNA Sequencing, Western Blot, Mass Spectrometry, Expressing, Software

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Normal B cell development in Igh Pax5/+ mice. (A–C) Flow-cytometric analysis of bone marrow (A), spleen (B), and lymph node (C) cells from 8–12-wk-old Igh Pax5/+ (black) and Igh +/+ (gray) littermates. The relative frequency was determined for pro-B (CD19 + Kit + CD2 – IgM – ), pre-B (CD19 + Kit – CD2 + IgM – ), immature B (CD19 + IgM + IgD – ), and recirculating B (CD19 + IgM lo IgD hi ) cells in the bone marrow (A), follicular (CD19 + CD21 lo CD23 hi ), and marginal zone (CD19 + CD21 hi CD23 lo ) B cells in the spleen (B) and mature CD4 + and CD8 + T cells in the lymph nodes (C). The flow-cytometric data were obtained in seven (A), five (B), or two (C) independent experiments. Statistical data (A–C) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P < 0.01; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot corresponds to one mouse. (D–F) Flow-cytometric analysis of Pax5 expression by intracellular staining of developing and recirculating B cells in the bone marrow (D), mature B cells in the spleen (E), and mature T cells in the lymph nodes of Igh Pax5/+ (black line) and Igh +/+ (gray filled) littermates. The specificity of the anti-Pax5 antibody (IgG2a) was controlled by staining of the different B cell types with a control IgG2a isotype antibody (gray dashed line).

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Two Tailed Test, Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Antibody secretion by Igh Pax5/+ plasma cells. (A and B) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells under steady-state conditions. The numbers of IgM and IgG ASCs in the bone marrow of Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) and 6–8 mo (B) were determined by ELISPOT assay by incubating RBC-depleted bone marrow cells (2 × 10 5 ) on IgM- or IgG-coated plates for 6 h before determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for IgM and IgG ASCs. (C and D) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells 14 d after immunization with NP-KLH (in alum). The numbers of anti–NP-IgM or anti–NP-IgG1 ASCs in the spleen (C) and bone marrow (D) were determined by ELISPOT assay by incubating RBC-depleted cells (2 × 10 6 ) from the spleen or bone marrow on NP 20 -BSA-coated plates before the determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. The data (C and D) are pooled from two independent immunization experiments. Statistical data (A–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot (A–D) corresponds to one mouse. (E) Electron microscope images of plasma cells isolated from the bone marrow of Igh Pax5/+ and Igh +/+ mice at the age of 6–8 mo. 1 representative image of 10 individual plasma cells analyzed per genotype is shown. The length of the indicated scale bars is 1 mm (B and D) or 0.5 µm (E).

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Enzyme-linked Immunospot, Two Tailed Test, Microscopy, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Antibody titers and morphology of plasma cells in Igh Pax5/+ mice. (A and B) Serum titers of total antibody of the IgM, IgG1, IgA, and IgE isotypes in nonimmunized Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) or 6–8 mo (B). The results shown are representative of three independent experiments. Statistical data (A and B) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05. NS, not significant (P > 0.05). (C) Expression of the indicated immunoglobulin isotype mRNAs in Igh Pax5/+ (black) and Igh +/+ (gray) plasma cells. The expression data are shown as mean TPM values with SD and are based on two ( Igh +/+ ) and three ( Igh Pax5/+ ) independent RNA-seq experiments. Each dot represents one experiment performed with pooled plasma cells from 5–10 mice per genotype. The P values were calculated by the DESeq2 program; ***, P < 0.001; ****, P < 0.0001. NS, not significant (P > 0.05). (D) Higher magnification of the same two electron microscope images shown in . The two plasma cells were isolated from the bone marrow of 6–8-mo-old Igh Pax5/+ and Igh +/+ mice. (E) Comparison of ER content in B cells and plasma cells. Staining with the ER-Tracker Red dye (at a final concentration of 0.2 µM) was used to determine the ER content of B cells (CD19 + ) and plasma cells (Blimp1-GFP + CD138 + ) in the bone marrow of 6–8-mo-old Igh Pax5/+ and Igh +/+ mice at steady state (left). The quantification of the staining is shown as mean fluorescence intensity (MFI; right). Statistical data (A, B, and E) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05. NS, not significant (P > 0.05). Each dot (A, B, and E) corresponds to one mouse.

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Two Tailed Test, Expressing, RNA Sequencing Assay, Microscopy, Isolation, Staining, Concentration Assay, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Rescue of IgG1 CSR in the presence of functional T cells. (A) Experimental strategy to interrogate B-lineage–intrinsic effects of ectopic Pax5 expression in response to immunization. CD43 – B cells isolated from the spleens of Igh B1-8hi/Pax5 Prdm1 Gfp/+ or Igh B1-8hi/+ Prdm1 Gfp/+ mice (CD45.1 + CD45.2 + ) were cotransferred with splenic T cells from OT-II TCR-tg Rag2 −/− mice (CD45.2 + ) into C57BL/6 recipients (CD45.1 + ). 1 d after cell transfer, the recipients were immunized with NP-OVA (in alum) and were analyzed 6 d after immunization. (B) Flow-cytometric analysis of splenic GC B cells of Igh B1-8hi donor origin. The frequencies of IgG1 + GC B cells of the Igh B1-8hi/+ Prdm1 Gfp/+ and Igh B1-8hi/Pax5 Prdm1 Gfp/+ genotypes were determined by sequential gating on donor cells (CD45.1 + CD45.2 + ), B cells (B220 + ), and GC B cells (GL7 + Fas + ) by flow cytometry (above). The quantification of the frequencies of GC B cells and IgG1 + GC B cells is shown below. The data are pooled from three independent experiments. (C) Analysis of splenic GFP + plasma cells derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ B cells. The flow-cytometric analysis is shown on the left, and the frequency of the GFP + plasma cells is quantified on the right. The data are pooled from two independent experiments. (D) ELISPOT assay to determine the frequency of anti–NP-IgM and anti–NP-IgG1 ASCs derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ cells. Six days after immunization with NP-OVA, 500 GFP + CD138 + plasma cells of donor origin were directly sorted onto NP 20 -BSA–coated plates. Cells were incubated for 6 h, before the number and size of anti–NP-IgM and anti–NP-IgG1 ELISPOTs were determined. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. Statistical data (B–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P < 0.01; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot (B–D) corresponds to one mouse. A scale bar of 1 mm length is shown.

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Functional Assay, Expressing, Isolation, Flow Cytometry, Derivative Assay, Enzyme-linked Immunospot, Incubation, Two Tailed Test

Journal: Molecular Cancer

Article Title: S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer

doi: 10.1186/s12943-022-01642-5

Figure Lengend Snippet: The key genes related to the innate resistance of CDK4/6 inhibitors were explored by analysis of ctDNA derived from patients with breast cancer. A - D Schematic overview of the study design and analytical workflow. PFS: progression-free survival. ctDNA: circulating tumour DNA. ddPCR: droplet digital PCR. IHC: immunohistochemistry. Multi-IF: multiplex immunofluorescence. E The landscape of high-frequency altered genes in plasma from the discovery patient cohort. The plot shows patients with innate resistance or clinical benefit following palbociclib treatment, with individual information about numbers of metastases, treatment lines, and endocrine therapy drugs used in combination with palbociclib. S6K1 (i.e. RPS6KB1) amplification was observed in three patients with innate resistance to palbociclib but not in those with clinical benefit

Article Snippet: The primary antibodies used in this study were as follows: S6K1 (Proteintech, Cat# 14485-1-AP), cyclin D1 (Proteintech, Cat# 60186-1-1 g), cyclin E1 (Abcam, Cat# ab33911), phospho-Rb (Abcam, Cat# ab109399), CDK2 (Proteintech, Cat# 10122-1-AP), CDK4 (Santa Cruz Biotechnology, Cat# sc-260), CDK6 (CST, Cat# 3136S), c-Myc (CST, Cat# 5605S), β-Actin (CST, Cat# 3700S), and GAPDH (Proteintech, Cat# 60004-1-Ig).

Techniques: Derivative Assay, Digital PCR, Immunohistochemistry, Multiplex Assay, Immunofluorescence, Clinical Proteomics, Amplification

Journal: Molecular Cancer

Article Title: S6K1 amplification confers innate resistance to CDK4/6 inhibitors through activating c-Myc pathway in patients with estrogen receptor-positive breast cancer

doi: 10.1186/s12943-022-01642-5

Figure Lengend Snippet: S6K1 promotes cell proliferation and palbociclib resistance via cell cycle progression. A Cell-cycle distribution was measured using PI staining followed by flow cytometry for MCF-7 cells transfected with S6K1 siRNA pool or non-sense control siRNA as well as exogenous S6K1-expressed T47D cells or control cells, respectively. P value was calculated by Student’s t -test. *, P < 0.05, **, P < 0.01. B , C KEGG pathway enrichment analysis ( B ) and GO functional analysis ( C ) of RNA-seq data obtained from S6K1-depleted MCF-7 cells. D GSEA showing an enrichment of E2F-targets and Myc-targets signatures in S6K1-depleted MCF-7 cells. E - G MCF-7 cells transfected with the indicated siRNAs as well as exogenous S6K1-expressed T47D cells or control cells with 70-80% confluent were harvested. Proteins were then subjected to western blotting with the antibodies against S6K1, cyclin D1, cyclin E1, CDK2, CDK4, CDK6, p-Rb, and c-Myc proteins. GAPDH/β-Actin was used as the loading control. H Dual luciferase reporter assay showing the transcriptional activity of CCNE1 promoter in S6K1-depleted MCF-7 cells. P value was calculated by Student’s t -test. **, P < 0.01. I c-Myc was re-expressed into S6K-depleted MCF-7 cells and the levels of cyclin E1 and p-Rb were measured by western blotting

Article Snippet: The primary antibodies used in this study were as follows: S6K1 (Proteintech, Cat# 14485-1-AP), cyclin D1 (Proteintech, Cat# 60186-1-1 g), cyclin E1 (Abcam, Cat# ab33911), phospho-Rb (Abcam, Cat# ab109399), CDK2 (Proteintech, Cat# 10122-1-AP), CDK4 (Santa Cruz Biotechnology, Cat# sc-260), CDK6 (CST, Cat# 3136S), c-Myc (CST, Cat# 5605S), β-Actin (CST, Cat# 3700S), and GAPDH (Proteintech, Cat# 60004-1-Ig).

Techniques: Staining, Flow Cytometry, Transfection, Control, Functional Assay, RNA Sequencing, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Summary of genome-wide STAT5 binding sites at L1. (A) The Venn diagram shows the number of identified STAT5A and STAT5B sites (peaks) in AABB tissue and STAT5B sites in BB tissue. (B) Average peak heights of STAT5A and STAT5B in AABB and BB tissues were estimated after library size normalization (RPM, reads per 10 million, input subtracted). (C) Mean fold changes of STAT5A and STAT5B target genes in AABB tissue and STAT5B target genes in BB tissue were calculated. The genes containing STAT5 peaks within ±1 kb around TSSs were regarded as STAT5 target genes. (D) Normalized tag counts (RPM) of STAT5A, RNA polII and H3K4me3 from 200 bp around STAT5A peak centers at L1 were calculated and compared between AABB and BB . Log 2 -transformed values were used ( x and y axes). (E) Normalized tags of H3K4me3 at positions 1 kb upstream and 2 kb downstream of TSS were summed up and divided by the size (3 kb) and then quantile normalized for comparison (top). The scatter plot shows the fold change ( x -axis) and difference ( y -axis) of H3K4me3 average enrichment between genes (spot) in AABB and BB . Cutoffs for significant changes were set as follows: 1.5-fold change ( x -axis, AABB/BB ) and four average tag difference ( y -axis, AABB/BB ). Among the genes showing significant changes of H3K4me3 enrichment, the number of STAT5 target and non-target genes was counted (bottom). (F) Genome browser views represent three gene loci ( Wap , Csn1s2a and Stap1 ) showing changes of H3K4me3 level and one housekeeping gene locus ( Actb ).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Genome Wide, Binding Assay, Transformation Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Histology and IF staining for NKCC1 of mammary tissues of mice with various STAT5 dosages in early pregnancy. ( A ) Transplanted tissues from mice expressing Stat5a or Stat5b at various levels as indicated were harvested on day 6 of pregnancy and stained with H&E. At this stage alveolar development in all samples is sparse in all epithelial cells expressing Stat5 and is even more reduced in Null cells (f). Black arrows indicate stromal adipocytes and white arrows indicate alveolar epithelial cells. Scale bar = 80 µm. ( B ) Staining of the membrane transporter molecule NKCC1, which is downregulated as epithelial cells differentiate, indicates a more mature developmental stage in wild type (a) cells, intermediate maturity in cells with two or one Stat5 alleles (b and c) and strong staining in Null cells (f). Arrowheads indicate NKCC1-positive cells stained in red. Myoepithelial cells are visualized with antibodies against smooth muscle actin (green).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Staining, Expressing, Membrane

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Histology and IF staining for NKCC1 of mammary tissues of mice with various STAT5 dosages at parturition. The nomenclature of mice with the different genotypes is based on the alleles they have retained. We refer to wild-type mice and Stat5ab fl/fl mice as AABB mice; Stat5ab fl/fl;MMTV- Cre (with Stat5ab -deficient mammary epithelial cells) as Null mice; Stat5a −/− mice as BB mice; Stat5b −/− mice as AA mice; Stat5ab +/ null mice as AB mice. Mice carrying only a single functional allele of either Stat5a ( Stat5ab null /Stat5b − ) or Stat5b ( Stat5ab null /Stat5a − ) are referred to as A mice and B mice, respectively. ( A ) Transplanted mammary tissues obtained from mice of different genotypes were collected on the day of parturition and analyzed by histology. Alveoli are expanded and filled with milk in the presence of four (a) and two (b and c) Stat5 alleles. Epithelial cells with only one active Stat5 allele (d and e) form dense alveoli lacking signs of secretory activity. Black arrows indicate stromal adipocytes and white arrows indicate alveolar epithelial cells. Scale bar = 80 µm. ( B ) Mammary tissues of transplanted epithelia obtained from mice of different genotypes were collected at parturition and sections were stained with anti-NKCC1 antibody (red) and α-smooth muscle actin (green). Arrowheads indicate NKCC1-positive cells stained in red. Myoepithelial cells are visualized with antibodies against smooth muscle actin (green).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Staining, Functional Assay, Activity Assay

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 regulation of and binding to known target genes

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 binding and chromatin features of the casein gene cluster. Genome browser tracks represent enrichment of STAT5A, STAT5B, H3K4me3 and RNA PolII in wild type ( AABB ) and Stat5a-null mammary tissues as well as STAT5 in liver and T cells. The liver and T-cell STAT5 ChIP-seq data sets were obtained from previous studies (GSE31578 and GSE36890). Conservation of GAS motifs (TTCnnnGAA) was calculated using the GERP score (the higher score means higher conservation) . Expression level of five milk protein genes was measured by both RNA-seq and qRT-PCR (bottom left). Absolute expression level of the milk protein genes is shown (bottom right).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, ChIP-sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 binding and chromatin features of STAT5 target genes. Genome browser tracks represent enrichment of STAT5A, STAT5B, H3K4me3 and RNA PolII in wild type ( AABB ) and Stat5a-null mammary tissues as well as STAT5 in liver and T cells. The liver and T-cell STAT5 ChIP-seq data sets were obtained from previous studies (GSE31578 and GSE36890). Conservation of GAS motifs (TTCnnnGAA) was calculated using the GERP score (the higher score means more conserved). ( A ) Wap gene, ( B ) Cish gene, ( C ) Socs2 gene, ( D ) Bcl6 gene and ( E ) Stat5a/b genes.

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, ChIP-sequencing

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: Leukemic progenitor assays replicate patterns of patient response to DRD2 antagonist TDZ (A) Leukemic blast counts were monitored before and after treatment with TDZ as a monotherapy in 11 relapsed or refractory AML patients (NCT02096289). Percentage change in blasts in the peripheral blood on day 5 versus day 1 is reported after treatment with TDZ. Percentage change in BM blast content is reported for trial patient 2T and 9T in the absence of circulating blast values. Partial response and progressive disease patterns are indicated as “response” and “no response” and are illustrated as gray versus black silhouettes, respectively. (B) Candidate trial patient samples from either response group were interrogated for progenitor content at baseline (day 1) and after clinical exposure to TDZ (day 5) using limiting dilution analysis (LDA). Leukemic progenitor frequency was estimated by LDA analysis and normalized to day 1. Baseline progenitor frequency of 1 in 75,000 cells was considered the progenitor frequency for trial patient 3T at day 1 since an absolute frequency was not achieved with the analysis of 75,000 cells for this patient. Dashed lines represent 95% confidence interval. Raw colony counts are shown in Figure S1 D. (C) Trial patient samples obtained at baseline were exposed to TDZ (“+TDZ”) versus DMSO control (“−TDZ”) for 24 h, followed by analysis of progenitor cell function in CFU assays. Data are normalized to DMSO control. Before normalization, the average DMSO control values were 79 and 2 colonies for trial patients 1T and 8T (non-responders) and 61, 28, 56, 2, 11, 28, and 14 colonies for trial patients 2T, 4T, 6T, 7T, 9T, 10T, and 11T, respectively (responders). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (D) Correlation between percentage change in leukemic blast levels versus percentage change in progenitor capacity (demonstrated in C). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (E) Schematic illustrating in vivo AML xenografts were treated with TDZ (22.5 mg/kg “+”) or 30% captisol (vehicle control “−”) in vivo , followed by analysis of leukemic chimerism levels (F), gene expression analysis (G), and progenitor CFU assays (H). (F) Leukemic chimerism levels (hCD45 + CD33 + ) after in vivo treatment with TDZ relative to vehicle control (“−“). Symbols represent individual recipient mice. ∗p = 0.05 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. (G) Gene set enrichment analysis (GSEA) plot of a gene set representing cellular pathways associated with AML (Kyoto Encyclopedia of Genes and Genomes [KEGG]; ), applied to transcription profiles from TDZ-treated versus vehicle control-treated AML xenografts derived from AMLs 1, 3, and 4. (H) Human AML grafts were recovered from mouse BM and evaluated in progenitor CFU assays. Symbols represent individual CFU wells, plated using cells recovered from a minimum of 2 individual mice per condition. Colony-forming capacity for AML 4 was not detectable with up to 150,000 human cells assayed. ∗∗∗p ≤ 0.0001 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. Data are summarized as means ± SEMs. See also and and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Control, Cell Function Assay, In Vivo, Gene Expression, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: DRD2 expression profiles reliably predict functional response to DRD antagonism (A) DRD2 expression patterns within leukemic CD34 + cells. Dotted line represents FMO control (left). Comparison of DRD2 protein levels in CD34 + cells of AML patient versus healthy donor samples (right). Healthy donor samples consist of cord blood (n = 3), adult mobilized peripheral blood (n = 3), and adult non-mobilized peripheral blood (n = 5). Blue versus red shading indicates the threshold of normal versus aberrant DRD2 levels. ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test). (B) DRD2 protein expression within CD34 + subset of low versus intermediate-/high-risk AML patients based on ELN criteria. Dots represent individual AML patients. ∗∗p = 0.006 (Mann-Whitney U test). (C) Mononuclear cells (MNCs) isolated from healthy donors and AML patients were treated with TDZ or DMSO (vehicle control, “−”) for 24 h and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n = 3–10 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). Source data can be found in . (D) Proliferative capacity of leukemic versus healthy progenitor units was compared after in vitro exposure to TDZ for 24 h. Cell number output per colony was evaluated by custom scripts as a measure of proliferation. (E) Representative FACS plots demonstrate gating strategy to purify DRD2 + vs DRD2 − human AML cells (left) and human leukemic chimerism in mice transplanted with 1 million DRD2 + or DRD2 − human AML cells. (F) Western blot of DRD2, activated CREB (p-CREB at Ser-133), and histone H3 (loading control) in DRD2 + versus DRD2 − sorted fractions illustrated in (E). (G) Representative whole-well CFU images after treatment with dopamine (DA) at physiological levels (10 nM) versus DMSO control (-DA). (H) Progenitor cell activity was quantified in n = 6 distinct AML patients after treatment with physiological levels of DA (10–100 nM) relative to DMSO control. n = 2–3 CFU wells per AML sample. ∗p = 0.03 (unpaired t test). (I) Circulating DA levels in healthy individuals (n = 8 healthy adult peripheral blood (PB) and 11 cord blood (CB) samples, as hollow circles and squares, respectively) versus n = 11 AML patients (black circles). ∗p = 0.04 (unpaired t test). Data are summarized as means ± SEMs relative to vehicle control. See also Figure S3 and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Expressing, Functional Assay, Control, Comparison, MANN-WHITNEY, Isolation, In Vitro, Western Blot, Activity Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: cAMP elevation is associated with leukemic progenitor suppression (A) Trial patients (NCT02096289) were exposed to TDZ in vitro , followed by analysis of cAMP level changes. Trial patients with abundant cell numbers available were prioritized for this analysis, including patients 1T and 3T from non-responders, and patients 7T, 10T, and 11T for responders. n = 3–6 technical replicates per condition. ∗p ≤ 0.05 (unpaired t test). (B) cAMP levels in response to DRD1 agonist (SKF 38393) relative to DMSO control. n ≥ 4 replicates across OCI-AML3 and NB4 cell lines. ∗∗p = 0.008 (Mann-Whitney U test). Progenitor response was evaluated after treatment with DRD1 agonist (SKF 38393) relative to DMSO control. n = 2–3 CFU replicates per AML sample (n = 5 AML samples total). (C) cAMP levels in response to anti-DRD1 antibody alone or in combination with DRD1 antagonist (SCH 23390) in AML cell lines OCI-AML3 and NB4. n = 2–4 replicates per condition. (D) Western blot of activated CREB (p-CREB at Ser-133) after exposure to anti-DRD1 antibody in OCI-AML3 cell line (top). Western blot of activated CREB (p-CREB at Ser-133) exposure to TDZ in OCI-AML3 and NB4 cell lines (bottom). (E) MNCs isolated from healthy donors and AML patients were treated with anti-DRD1 antibody or immunoglobulin G (IgG) control (“−“) for 30 min, and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n =3–7 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). (F) Cytospin preparations of AML cells from patient 2 after exposure to TDZ or vehicle control (DMSO). Yellow arrowheads indicate evidence of hematopoietic maturation (increased cell size, reduced nuclear:cytoplasmic ratio, increased cytoplasmic vacuolization). (G) FACS plot showing expression of granulocytic cell marker (CD15) after in vitro exposure to TDZ or DMSO control (“-TDZ“) in representative DRD2 lo and DRD2 + AML samples. CD15 frequencies were quantified for AMLs 1, 6, and 7 (n = 2 technical replicates per AML sample in each condition). ∗∗p = 0.002 (Mann-Whitney U test). (H) AML patient cells were treated with TDZ or DMSO for 24 h and evaluated in progenitor CFU assays, followed by analysis of re-plating capacity. ∗∗p = 0.004 (unpaired t test). (I) cAMP levels in response to TDZ relative to DMSO control. DRD2 + AML includes AML 1, 6, OCI-AML3, and NB4. DRD2 − AML and healthy controls include AML 12 and 3 CB samples, respectively. n ≥ 3 replicates per condition. ∗∗∗p = 0.007 (unpaired t test). (J) cAMP levels in response to forskolin (FSK) relative to DMSO control. n = 6 replicates per condition, across 1 AML cell line and n = 2 healthy donor cells. ∗∗∗p ≤ 0.0001 (unpaired t test). Data are summarized as means ± SEMs. See also Figure S4 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: In Vitro, Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: TDZ + displays superior potency and reduced toxicity relative to TDZ (A) Chiral separation of TDZ using supercritical fluid chromatography. Chromatograms show the first and second peaks, indicating the (−) enantiomer “TDZ − ” and (+) enantiomer “TDZ + ,” respectively. Purified enantiomers were evaluated for effects on cAMP levels (B), and in progenitor CFU assays (C and D). (B) cAMP levels were evaluated after in vitro treatment with TDZ and its two enantiomers in AML cell lines (NB4 and OCI-AML3) and primary patient cells (AMLs 2, 9, and 27). Symbols represent individual CFU wells. ∗p ≤ 0.05 and ∗∗p ≤ 0.01 (unpaired t test). (C) AML patient cells were exposed to TDZ and its 2 enantiomers for 24 h in a dose-response assay in vitro , and subsequently evaluated in progenitor CFU assays. Bar graphs summarize half-maximal inhibitory concentration (IC 50 ) in progenitor CFU assays performed with AML patient cells. ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001 (paired t test). (D) Comparison of TDZ and TDZ + IC 50 for individual AML patients in CFU assays (represented in C). ∗∗p = 0.004 (paired t test). (E) A 30-min monitoring of QTc level changes after intravenous injection of TDZ and TDZ + in a guinea pig assay (n = 5 animals per cohort). QTc increases over 5% were considered indicators of safety risks. No group averages were statistically different from baseline values (repeated-measures ANOVAs). (F) DRD2 transcript (Gene: 1813) was analyzed from TGCA (tumor and normal tissue) and GTEx (normal tissue) RNA-sequencing projects. Data points represent normalized gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) for DRD2 from individual cancer patients or healthy donors. ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test), ∗∗p = 0.01 (Kolmogorov-Smirnov test). Data are summarized as means ± SEMs. See also Figure S5 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Supercritical Fluid Chromatography, Purification, In Vitro, Concentration Assay, Comparison, Injection, RNA Sequencing, Gene Expression, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet:

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Recombinant, Binding Assay, Purification, Microarray, Software, Imaging

Journal: Genes & Development

Article Title: Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

doi: 10.1101/gad.216804.113

Figure Lengend Snippet: Identification of active enhancers in quiescent NS cells. ( A ) Heat map representation of the density of ChIP-seq reads for H3K27ac and p300 ±2 kb relative to the midpoint of enriched regions at 16,246 active enhancers in NS cells. This panel represents the merger of data obtained in proliferating and quiescent NS cells. A large fraction of the regions displayed presents active enhancer features only in proliferating NS cells or only in quiescent NS cells, and a smaller fraction presents these features in both cellular states. Intensity of color represents the normalized statistical significance of the signal versus input control sequences. ( B , C ) H3K27ac and p300 ChIP-seq signal and RNA expression level (FPKM) in quiescent (blue) and proliferating (green) NS cells in the vicinity of Id4 and Vash1, two representative genes that are up-regulated in quiescent and proliferating NS cells, respectively. Regions defined as quiescent and proliferating NS cell-specific enhancers are indicated by blue and green rectangles, respectively. ChIP-seq peak height corresponds to SICER P -value for H3K27ac and MACS Q -value for p300. ( D ) Average ChIP-seq signal profile for H3K27ac in quiescent (blue line) and proliferating (green line) NS cells and several other epigenetic marks in proliferating NS cells at regions defined as quiescent ( left ) and proliferating ( right ) NS cell-specific enhancers. Plots are centered on the p300 summit. Quiescent NS cell-specific enhancers show strong signals for the enhancer-associated H3K4me1 mark and weak signals for the open chromatin-associated H3K4me2 and H3K27ac marks in proliferating NS cells, consistent with these regions being marked as enhancers but minimally active in proliferating NS cells. Proliferating NS cell-specific enhancers have strong signals for H3K27ac, H3K4me1, and H3K4me2 but not the other nonenhancer-associated epigenetic modifications. Note the dip in the enrichment profile for H3K27ac, indicative of a localized depletion of nucleosomes characteristic of enhancers ( ; ). ( E ) Box plots of normalized transcript counts (FPKM) for all genes expressed in quiescent NS cells ( left ) and genes associated with quiescent NS cell-specific enhancers ( right ). The latter are expressed at higher levels than the transcriptomic average (Wilcoxon test, P < 2.2 × 10 −16 ). ( F ) Fraction of genes up-regulated in quiescent NS cells whose closest enhancer is quiescent NS cell-specific ( left ), pan-NS cell ( middle ), or proliferating NS cell-specific ( right ). Asterisk denotes significant P -value (Wilcoxon test). See also Supplemental Figure S3.

Article Snippet: Immunoprecipitations were with rabbit anti-H3K27ac (4 μg per ChIP sample; Abcam, ab4729), rabbit anti-p300 (3 μg per ChIP sample; Santa Cruz Biotechnology, sc-585), or goat anti-NFI (6 μg per ChIP sample; Santa Cruz Biotechnology, sc-30918).

Techniques: ChIP-sequencing, Control, RNA Expression

Journal: Genes & Development

Article Title: Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

doi: 10.1101/gad.216804.113

Figure Lengend Snippet: The NFI motif is most strongly overrepresented in quiescent NS cell enhancers. ( A – C ) DNA sequence motifs matching consensus binding sites for NFI ( A ), Sox ( B ), and bHLH ( C , E-box) TFs are found overrepresented in quiescence-specific, pan-NS cell, and activity-specific enhancers by de novo motif searches. ( D ) Enrichment values ( E -values) of NFI, Sox, and E-box motifs in quiescence-specific, pan-NS cell-specific, and activity-specific enhancers as reported by DREME. ( E – G ) Observed frequency of motif occurrence around the summit of p300 binding in quiescence-specific, pan-NS cell, and activity-specific enhancers. The NFI motif is the most overrepresented in quiescence-specific and pan-NS cell enhancers. ( H ) RNA-seq shows that all four NFI genes are transcribed in NS cells, with transcript levels of Nfix increasing sharply in quiescent cells, while those of Nfia , Nfib , and Nfic decrease or remain unchanged. ( I , J ) Immunocytochemistry shows that NFIX protein is strongly induced in quiescent NS cells, while NFIA expression is reduced. See also Supplemental Figure S4.

Article Snippet: Immunoprecipitations were with rabbit anti-H3K27ac (4 μg per ChIP sample; Abcam, ab4729), rabbit anti-p300 (3 μg per ChIP sample; Santa Cruz Biotechnology, sc-585), or goat anti-NFI (6 μg per ChIP sample; Santa Cruz Biotechnology, sc-30918).

Techniques: Sequencing, Binding Assay, Activity Assay, RNA Sequencing, Immunocytochemistry, Expressing

Journal: Genes & Development

Article Title: Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

doi: 10.1101/gad.216804.113

Figure Lengend Snippet: NFI TFs bind to the majority of quiescent NS cell enhancers. ( A ) Heat map representation of all enhancers active in quiescent NS cells sorted into quiescent-specific and pan-NS cell enhancers showing ChIP-seq signal for NFI TFs, H3K27ac, and p300. ( B ) Venn diagram showing the large overlap of enhancers in quiescent NS cells with regions of significant NFI TF binding. ( C , D ) Strong correlation of the strength of ChIP-seq signals for p300 and NFI in enhancers ( C ; correlation coefficient = 0.67) and close proximity of their summits ( D ; median intersummit distance = 35 base pairs [bp]), consistent with p300 recruitment by NFI TFs. ( E , F ) Functional annotation of quiescence-specific ( E ) and pan-NS cell ( F ) enhancers bound by NFI TFs by GREAT according to GO biological process. Enhancers bound by a NFI factor (purple) and those that are not significantly bound (green) were examined separately. The X -axis values represent the binomial FDR Q -values; the numbers in parentheses are the number of binomial region hits.

Article Snippet: Immunoprecipitations were with rabbit anti-H3K27ac (4 μg per ChIP sample; Abcam, ab4729), rabbit anti-p300 (3 μg per ChIP sample; Santa Cruz Biotechnology, sc-585), or goat anti-NFI (6 μg per ChIP sample; Santa Cruz Biotechnology, sc-30918).

Techniques: ChIP-sequencing, Binding Assay, Functional Assay

Journal: Genes & Development

Article Title: Epigenomic enhancer annotation reveals a key role for NFIX in neural stem cell quiescence

doi: 10.1101/gad.216804.113

Figure Lengend Snippet: NFIX is both required and sufficient to induce aspects of quiescence in NS cell cultures. ( A ) Efficiency of Nfix silencing in NS cells exposed to BMP to induce quiescence at the time of shRNA electroporation analyzed by qPCR 1, 2, and 3 d after shRNA transfection and BMP exposure. A scrambled shRNA was used in the control experiment, and expression of the gene ActB is analyzed for comparison. Note that Nfix transcript levels increase progressively between days 1 and 3 as cells enter quiescence in both control and Nfix knockdown experiments. ( B ) Analysis of proliferation by EdU immunostaining after 4 h of exposure in NS cell cultures following 1, 2, and 3 d of BMP exposure as indicated. Cells are counterstained with DAPI (blue). ( C ) Percentages of EdU-positive NS cells in Nfix shRNA transfected and control cultures. The BMP-induced cell cycle arrest is delayed by Nfix silencing. The progressive reduction in cell proliferation of Nfix shRNA-treated cultures between days 1 and 3 might be due to the progressive increase in Nfix expression during this period (shown in A ). Error bars represent the standard deviation ( n = 3 biological replicates). ( D ) Analysis of proliferation by EdU immunostaining in NS cell cultures transfected 18 h earlier with a Nfix expression construct and GFP or with GFP alone. ( E ) Percentages of EdU-positive cells in NS cell cultures transfected with GFP or GFP and Nfix . Nfix efficiently promotes cell cycle arrest. ( F ) Venn diagram showing the large fraction of genes regulated in quiescent NS cells that are also regulated by Nfix . GO analysis of Nfix-activated genes that are also induced in quiescent NS cells ( G ) and Nfix-repressed genes that are up-regulated in proliferating NS cells ( H ). ( I ) Representative examples of putative NFI direct target genes (associated with a NFI-bound enhancer/promoter and activated by Nfix ) induced in quiescent NS cells and belonging to functionally important GO categories. ( J ) ChIP-seq signal for H3K27ac, p300, and NFI and RNA-seq signal (FPKM) for Svep1 and Bgn , two representative NFI direct target genes up-regulated in quiescent NSCs. Significant NFI binding within enhancer regions is indicated by pale blue rectangles. Peak height corresponds to SICER P -value for H3K27ac and MACs Q -value for p300 and NFI. See also Supplemental Figure S5 and Supplemental Table S2.

Article Snippet: Immunoprecipitations were with rabbit anti-H3K27ac (4 μg per ChIP sample; Abcam, ab4729), rabbit anti-p300 (3 μg per ChIP sample; Santa Cruz Biotechnology, sc-585), or goat anti-NFI (6 μg per ChIP sample; Santa Cruz Biotechnology, sc-30918).

Techniques: shRNA, Electroporation, Transfection, Control, Expressing, Comparison, Knockdown, Immunostaining, Standard Deviation, Construct, ChIP-sequencing, RNA Sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

doi: 10.1093/nar/gkv695

Figure Lengend Snippet: Mutational analysis of Ty1i RNA initiation codons AUG1 and AUG2. ( A ) Map of the Ty1 and pGTy1 mutant derivatives used to assess translation of Ty1i RNA from two closely spaced initiation codons AUG1 and AUG2. pG POL Δ lacks most of POL , but contains GAG , including sequences required for transcribing Ty1i RNA and cleavage of p22 to p18 by Ty1 protease (PR). ( B ) pG POL Δ ( URA3 , 2μ) plasmids were introduced into a Ty1-less Saccharomyces paradoxus strain containing a single chromosomal Ty1 his3-AI element. Cells propagated on glucose are repressed for transcription of Ty1 mRNA from GAL1 promoter, but allow synthesis of Ty1i RNA and p22/p18. Ty1 his3-AI mobility analyses using this assay are shown in Table . ( C ) Total cell protein isolated after growth in SC-Ura medium for 2 days at 22°C was immunoblotted with p18 antiserum (anti-p18). Histidyl tRNA synthetase (anti-Hts1) served as a loading control.

Article Snippet: To determine if the CTR interacts with p18, GST-complexes were analyzed by immunoblotting using mouse monoclonal antibody GST/B-14 (Santa Cruz Biotech) or rabbit polyclonal p18 antibody ( ).

Techniques: Mutagenesis, Isolation, Control

Journal: Nucleic Acids Research

Article Title: Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

doi: 10.1093/nar/gkv695

Figure Lengend Snippet: Effect of endogenous p22/p18 expression on Ty1 his3-AI mobility

Article Snippet: To determine if the CTR interacts with p18, GST-complexes were analyzed by immunoblotting using mouse monoclonal antibody GST/B-14 (Santa Cruz Biotech) or rabbit polyclonal p18 antibody ( ).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

doi: 10.1093/nar/gkv695

Figure Lengend Snippet: Functional organization of GAG and coexpression of subgenomic segments with pGTy1 his3-AI . ( A ) At the top is the mature Gag (p45) coding sequence with selected ATG codons highlighted (green) and below are segments expressed ectopically from the pYES2 GAL1 promoter. At the bottom, XtalPred ( http://ffas.burnham.org/XtalPred-cgi/xtal.pl ) was used to predict Gag disordered (yellow) and α-helical (red) regions. Also shown is an invariant tryptophan residue (W184) found in Pseudoviridae Gag proteins , the position of the nucleic acid chaperone region , a C-terminal disordered region (C-DR), and the Ty1 protease (PR) cleavage site (H401-N402). An ATG codon was added adjacent to P173 for expression of the CTR and sCTR. ( B ) Two plasmids, pGTy1 his3-AI ( TRP1 , CEN) and GAG segment under pYES2 ( URA3 , 2 μ) were induced from the GAL1 promoter in a Ty1-less Saccharomyces paradoxus strain to determine whether different Gag proteins inhibited Ty1 his3-AI mobility (Table ). ( C ) Total cell protein from induced cultures was immunoblotted with p18 antiserum (anti-p18) or TY-tag (anti-TY-tag). Histidyl tRNA synthetase (anti-Hts1) served as a loading control.

Article Snippet: To determine if the CTR interacts with p18, GST-complexes were analyzed by immunoblotting using mouse monoclonal antibody GST/B-14 (Santa Cruz Biotech) or rabbit polyclonal p18 antibody ( ).

Techniques: Functional Assay, Sequencing, Residue, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

doi: 10.1093/nar/gkv695

Figure Lengend Snippet: RNA binding properties of CTR, AUG2p18 and AUG1p18 proteins. Data plots of the filter-binding assay performed in different concentrations of NaCl (10–500 mM) for Ty1 mini RNA and ( A ) CTR, ( B ) AUG2p18 and ( C ) AUG1p18. The lines correspond to the best fit of the data. ( D ) Data plot of the dissociation constant measured for CTR, AUG2p18 and AUG1p18 as a function of NaCl concentration. The error bars represent standard deviations. ( E ) 2D structure model of +1–362 region of Ty1 mini RNA with the positions protected from hydroxyl radical cleavage in the presence of the Ty1 Gag derived proteins marked (red). ( F ) Reactivity plots of protein free Ty1 mini RNA (black) in comparison with RNA probed in the presence of CTR (red) and p18 (green). Regions showing consistent increased reactivity over several nucleotides are boxed.

Article Snippet: To determine if the CTR interacts with p18, GST-complexes were analyzed by immunoblotting using mouse monoclonal antibody GST/B-14 (Santa Cruz Biotech) or rabbit polyclonal p18 antibody ( ).

Techniques: RNA Binding Assay, Filter-binding Assay, Concentration Assay, Derivative Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

doi: 10.1093/nar/gkv695

Figure Lengend Snippet: GST-CTR interacts with p18 or deleted CD-R p18. Protein extracts (Input) from a Ty1-less strain induced for expression of GST [pEG(KT); pGST], GST-CTR (pBDG1496; pGST-CTR) and ( A ) p18 (pAUG1p18) or ( B ) p18 lacking the C-DR (pBDG1612; psAUG1) were incubated with glutathione-coated resin. Bound proteins (Pull-down) were analyzed by immunoblotting with GST and p18 antisera to detect, GST, GST-CTR, p18 or deleted C-DR p18 after extensive washing with lysis buffer. A putative p18 degradation product of ∼12.5 kDa is noted in panel A (*; also refer to Supplementary Figure S7).

Article Snippet: To determine if the CTR interacts with p18, GST-complexes were analyzed by immunoblotting using mouse monoclonal antibody GST/B-14 (Santa Cruz Biotech) or rabbit polyclonal p18 antibody ( ).

Techniques: Expressing, Incubation, Western Blot, Lysis

Journal: Nucleic Acids Research

Article Title: Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

doi: 10.1093/nar/gkv695

Figure Lengend Snippet: Impact of p22/p18 or p18 on RNA dimerization and packaging. ( A ) A representative electrophoretic analysis in the presence of increasing concentrations of CTR, AUG2p18 or AUG1p18. Proteins concentrations correspond to 1:20; 1:10; 1:7 and 1:5 protein to nt ratios. Lanes denoted C represent control samples that lack protein. ( B ) The graph representing the averaged percent of Ty1 mini RNA dimer from three independent experiments. The error bars represent standard deviations. ( C ) Nuclease protection of Ty1 mRNA in cells coexpressing pGTy1 his3-AI alone and pAUG1p22 or pAUG1p18. Equal aliquots of whole-cell extracts from galactose-induced cells were incubated with (+) or without (−) the nuclease benzonase. RNA extracted from these samples was analyzed by Northern blotting using a 32 P-labeled riboprobe specific for Ty1 mRNA. Protection from benzonase is expressed as a ratio of treated to untreated Ty1 mRNA. ACT1 was used as a control to confirm RNA degradation in the nuclease treated samples. ( D ) Total protein extracts used in (C) was immunoblotted with p18 antiserum (anti-p18). Histidyl tRNA synthetase (anti-Hts1) served as a loading control.

Article Snippet: To determine if the CTR interacts with p18, GST-complexes were analyzed by immunoblotting using mouse monoclonal antibody GST/B-14 (Santa Cruz Biotech) or rabbit polyclonal p18 antibody ( ).

Techniques: Control, Incubation, Northern Blot, Labeling

Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: (A) Diagram of ASH1L protein domains showing the location of the pathogenic variant E2148* (blue) in ASH1L catalytic domain and itsr associated clinical phenotypes. (B) Illustration depicts the dual SMAD inhibition protocol used to generate cortical excitatory human neurons. ( C ) ASH1L expression was quantified by qPCR using human neurons at day 35 of neuronal induction. Fold change is normalized to control. Bar represents the mean and individual measures from four independent experiments are shown for control (grey with open circles), and E2148* (light blue with solid blue circles). Samples were analyzed as a ratio of the control. Statistical analysis was conducted using unpaired t-test. **** P < 0.0001. ( D ) Representative images are shown for human neurons from control, and E2148* cultures at day 35 of neuronal induction. Neurons stained with MAP2 are shown in black and white for ease of viewing. Calibration bars represent 20µm. ( E-H ) Morphogenesis measures are shown for four independent experiments for control neurons (grey bar with open circles), and E2148* mutant neurons (light blue bars with solid dark blue circles). Individual points represent the average of 4 independent experiments, an average of 30 neurons were measured per experiment. ( E ) Mean neurite length is shown for control (n=124 neurons; 56.9 ± 2.41), and E2148* (n=118 neurons; 47.47 ± 1.99). Grouped statistical analysis was conducted using unpaired t-test, **P < 0.004. ( F ) Total neurite length is shown for control (n=124 neurons; 182.7 ± 6.39), and E2148* (n=118 neurons; 139.3 ± 4.66). Grouped statistical analysis was conducted using unpaired t-test, **** P < 0.0001. ( G ) Neuronal morphology analyzed by measuring the complexity index (see methods). Calculations were conducted after identifying outliers using the ROUT 1% method for control (n=115; 289.5 ± 18.21), and E2148* (n=112; 228.8 ± 13.42). Grouped statistical analysis was conducted unpaired t-test ** P < 0.0099. ( H ) Cell soma size was analyzed for three independent experiments by measuring the area for control (n=96; 77.67 ± 3.47), and E2148* (n=91; 69.15 ± 2.51). Statistical analysis was conducted using unpaired t-test P=0.056. ( I ) Sholl analysis was used to measure neuronal arborization. The number intersections away from the cell soma were measured every 10µm and are shown for control (open gray circles), and E2148* (solid dark blue circles) neurons from 10µm to 120µm. Statistical analysis was conducted using a mixed model effects *** P < 0.0006, and **** P < 0.0001. ( J-L ) Analysis of H3K36me2 and H3K4me3 levels on chromatin fraction for four independent experiments is shown for neurons at day 41 of neuronal induction. ( J ) Representative western blot shows H3K36me2, H3K4me3 and histone H3 for control, and E2148* neurons. H3 Histone marks were normalized to histone H3 levels for analysis. ( K ) H3K36me2 protein levels are shown for control (1± 0), and E2148* (0.67 ± 0.26). ( L ) H3K4me3 protein levels are shown for control (1± 0), and E2148* (0.68± 0.11). (K -L ) Statistical analysis was conducted using unpaired t-test *P< 0.025. Not significant P value is not shown.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Variant Assay, Inhibition, Expressing, Control, Staining, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: ( A ) PCA plots shows biological replicates (n=4) for control (green), and E2148* (salmon) neurons RNA seq experiments. ( B ) Heatmap shows top 100 DEGs for control (green), and E2148* (salmon) neurons at day 35 (n=4 biological replicates). The top 15 DEGs are listed. ( C ) Volcano plots showing DEGs in the heterozygous E2148* mutant iPSC-derived neurons. Log 2 fold changes (LFC) gene expression (x-axis) and -log 10 adjusted P values (y-axis) generated from DESeq2 differential gene expression analysis are shown. Vertical dotted lines represent 0.58 LFC (1.5 FC) and horizontal dotted line shows adjusted P=0.05. Significant DEGs are shown in red with the top 20 labelled in the plot. ( D-F ) Functional enrichment analysis by EnrichR for biological process ( D ), cellular compartment ( E ), and molecular function ( F ) show enrichment for all DEGs, upregulated and downregulated DEGs in E2148* mutant neurons vs. control neurons. Circle size represents the number of DEGs in that category and the color represents the adjusted P value. ( G ) Correlation of gene length to fold change analyzed for all significant DEGs in E2148* (blue line) mutant neurons. Grey shade shows the variability across samples. ( H ) Analysis of gene length in upregulated (blue) and downregulated (red) DEGs for E2148* neurons. ( I ) Analysis of de novo transcription by EU click chemistry at day 41 of neuronal differentiation. Representative images of human neurons that incorporated EU (gray), stained with neuronal marker MAP2 (cyan) and nuclear marker DAPI (blue) are shown for control (top row), and E2148* (bottom row). Enlarged nuclei stained with EU is shown. Calibration bars are 20µm. ( J-K ) Measurements of EU incorporation are shown for control neurons (grey bars with open circles), and E2148* (light blue bars with solid deep blue circles) mutant neurons. Mean and standard error are shown with individual dots representing the average of individual measures for five independent experiments. ( I ) Pearsons’ correlation coefficient analysis is shown for five independent experiments for control (n=187; 0.785 ± 0.003), and E2148* (n=115; 0.746 ± 0.005) neurons. ( J ) EU nuclear intensity normalized to control is shown for five independent experiments for control (n=187; 1.017 ± 0.029), and E2148* (n=115; 0.817 ± 0.027) neurons. ( I-J ) Grouped data analyzed using unpaired t test with Welch’s correction, ****P < 0.0001. Not significant P values are not shown.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Control, RNA Sequencing, Mutagenesis, Derivative Assay, Gene Expression, Generated, Functional Assay, Staining, Marker

Journal: bioRxiv

Article Title: Dynamic Regulation OF The Chromatin Environment By Ash1L Modulates Human Neuronal Structure And Function

doi: 10.1101/2024.12.02.625500

Figure Lengend Snippet: (A) Representative images are shown for day 35 human neurons from control, and E2148* cultures treated for 3 days with DMSO, Tazemetostat (0.5µM) and Vorinostat (0.1µM). Neurons stained with MAP2 are shown in black and white for ease of viewing. Calibration bars represent 30µm. ( B-F ) Morphogenesis analysis is shown for at least 4 independent experiments (unless otherwise annotated) in which we measured at least 30 neurons per experiment for control (grey bar with open circles) and E2148* (light blue bars with solid dark blue circles) neurons treated with either DMSO, Tazemetostat (TAZ) or Vorinostat (VOR). Individual points represent the average of multiple independent experiments. ( B ) Total neurite length is shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=93 neurons; 231.9 ± 7.27); control + TAZ (n=119; 184.4± 5.46); control + VOR (n=118 neurons; 227.3± 6.8); E2148* + DMSO (n=113 neurons; 163.3 ± 4.83); E2148* + TAZ (n=112; 200.5± 5.94); E2148* + VOR (n=129 neurons; 225.2± 8.38). ( C ) Mean neurite length is shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=90 neurons; 69.86 ± 2.409); control + TAZ (n=117; 64.16± 2.04); control + VOR (n=118 neurons; 81.28± 2.75); E2148* + DMSO (n=112 neurons; 55.75 ± 1.88); E2148* + TAZ (n=111; 66.16 ± 2.47); E2148* + VOR (n=126 neurons; 76.02 ± 2.58). ( D ) Complexity index measurements were first analyzed using the “identify outliers” ROUT function in graph pad and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=90 neurons; 390± 26.88); control + TAZ (n=111; 329.4 ± 22.40); control + VOR (n=116 neurons; 461.8 ± 25.45); E2148* + DMSO (n=116 neurons; 261.9 ± 18.80); E2148* + TAZ (n=105; 357.6 ± 20.83); E2148* + VOR (n=125 neurons; 458.3 ± 29.66). ( B-D ) Statistical analysis of grouped measurements was conducted using TWO-way ANOVA with Tukey’s test for multiple comparisons: * P < 0.04 ** P < 0.009, *** P < 0.0006, **** P < 0.0001. ( E ) Sholl analysis was used to measure neuronal arborization across three different treatments in the E2148* mutant neurons. The number intersections away from the cell soma were measured every 10µm and are shown for E2148* + DMSO (inverted dark blue triangles), E2148* + TAZ (open triangles), and E2148* + VOR (solid light blue triangles) neurons. Statistical analysis by TWO-way ANOVA with mixed model effects * P < 0.05, ** P < 0.009, and *** P = 0.0008. Green asterisk (E2148* +DMSO vs. E2148* + VOR), red asterisk (E2148* + DMSO vs. E2148* + TAZ). ( F ) Sholl analysis is shown to compare the most effective treatment (vorinostat) to the untreated control and E2148* mutant neurons. The number of intersections away from the cell soma were measured every 10µm and are shown for control+ DMSO (open gray circles), E2148* + DMSO (solid dark blue circles) and E2148* + VOR (half pink/light blue circles) neurons. Statistical analysis by TWO-way ANOVA with mixed model effects * P < 0.05, ** P < 0.005, *** P = 0.0005, and **** P < 0.0001. Green asterisk (E2148*+DMSO vs. Control + DMSO), red asterisk (E2148* + DMSO vs. E2148* + VOR). ( G-K ) Analysis of nuclear levels of H3K27me3 and H4K16ac in four independent experiments (unless otherwise indicated) across all treatments is shown for neurons at day 35 of neuronal induction. ( G ) Representative images of nuclear H3K27me3 (red) are shown for either DMSO (left column) or Tazemetostat (right column) treated control, or E2148* mutant neurons stained with MAP2 (cyan) and nuclei is stain with DAPI (blue). ( H ) Quantification of H3K27me3 nuclear levels measured by mean gray value is shown for all treatments. Measurements from at least 3 independent experiments with at least 30 neurons analyzed per experiment were analyzed as a group and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (n=134 neurons; 547.4 ± 13.19); control + TAZ (n=154; 258.1 ± 10.83); control + VOR (n=101 neurons; 545.5 ± 19.07); E2148* + DMSO (n=141 neurons; 471.0 ± 10.87); E2148* + TAZ (n=140; 301.1 ± 8.03); E2148* + VOR (n=103 neurons; 539.4 ± 18.79). ( I ) Representative images of nuclear H4K16ac (red) are shown for either DMSO (left column) or Tazemetostat (right column) treated control, and E2148* mutant neurons stained with MAP2 (cyan) and nuclei is stain with DAPI (blue). ( J ) Quantification of H4K16ac nuclear levels measured by mean gray value is shown for all treatments. Measurements from at least 3 independent experiments with at least 30 neurons analyzed per experiment were analyzed as a group and are shown as the mean (bar) with the average of individual measurements represented by the circles for: control + DMSO (N= 4 experiments; n=114 neurons; 322.8 ± 12.85); control + TAZ (n=95; 360.5 ± 13.04); control + VOR (n=145 neurons; 581.6 ± 16.45); E2148* + DMSO (n=166 neurons; 270.3 ± 10.33); E2148* + TAZ (n=106; 350.4 ± 11.81); E2148* + VOR (n=158 neurons; 654.7 ± 9.49). ( H and J ) Statistical analysis of grouped measurements was conducted using TWO-way ANOVA with Tukey’s test for multiple comparisons: * P < 0.05, *** P < 0.005, *** P < 0.0005, **** P < 0.0001.

Article Snippet: Primary antibody for MAP2 (Aves lab, #MAP) was added and cells were incubated overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature using Alexa Fluor 647 (Thermo Scientific, #A-21449) at a dilution of 1:1000.

Techniques: Control, Staining, Mutagenesis