Journal: Nature medicine

Article Title: Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia

doi: 10.1038/nm.3543

Figure Lengend Snippet: Cardiomyocyte T-tubules are densely folded by BIN1. ( α–b ) Representative confocal images ( a , scale bars: 5 µm) and the fluorescent profiles ( b ) of live WT and Bin1 HT cardiomyocytes labeled with Di-8-ANNEPS. ( c ) Quantification of T-tubules peak intensity. ( n = 40 from 4–5 cells, P < 0.0001). ( d ) Cell size normalized membrane capacitance in WT ( n = 14) and Bin1 HT ( n = 12) cardiomyocytes ( P = 0.0181). WC indicates reported whole cell capacitance without T-tubules. ( e ) 2D transmission electron microscope (TEM) images (Left to right: gross morphology, transverse cross section, and axial cross section) and 3D electron tomography images (right) of WT and Bin1 HT heart sections. Scale bars (left to right): 1 µm, 250 nm, 100 nm, and 100 nm. ( f ) Electron density profiles (middle) across individual T-tubules marked by the lines in the images above, with average T-tubule electron density in the bottom ( n = 75, P < 0.0001). ( g ) T-tubule lumen area of axial cross sections ( n = 80, P < 0.0001). ( h ) Cardiomyocyte T-tubule contour score (1, circular shape and no folds and spatial complexity; 2, non-circular shape and no folds and spatial complexity; or 3, multiple folds with spatial complexity) distribution ( n = 196, P < 0.0001). Data are presented as mean ± SEM, cardiomyocytes are from three mice per genotype, and six left ventricular sections from three hearts per genotype were used for TEM analysis. Student’s t -test and one way-ANOVA were used for statistical analysis.

Article Snippet: To confirm the efficiency of Cre-mediated deletion of Bin1 , quantitative real-time PCR using TaqMan primer/probe sets (5'FAM/3'BHQ; Applied Biosystems) spanning constitutively splice exons 2 and 3 (Mm01158690_mH) was used to measure transcription of total Bin1 gene, the house-keeping gene Hrpt1 , and a cardiac specific gene TnI3 in cardiomyocyte cDNA generated from both WT and Bin1 HT mice.

Techniques: Labeling, Membrane, Transmission Assay, Microscopy, Tomography

Journal: Nature medicine

Article Title: Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia

doi: 10.1038/nm.3543

Figure Lengend Snippet: Bin1 deletion increases extracellular Ca 2+ diffusion. ( a ) Representative patch clamp recording of the LTCC mediated I Ca from a WT cardiomyocyte in response to quick change from 2 mM extracellular calcium solution to calcium free 5 mM EGTA solution. ( b ) Kinetics of I Ca current changes using the protocol described in ( a ) were fitted with one plateau followed by one phase exponential decay. X 0 is the initial delay before I Ca decays. ( c ) Comparison of X 0 for WT and Bin1 HT. Data are presented as mean ± SEM, P = 0.0001 by student’s t -test (cardiomyocytes are from 3 mice for each genotype). ( d ) A diagram describing the salient features of a mathematical model for calcium diffusion. ( e ) Kinetics of I Ca current decay computed using the model in ( d ). The normalized calcium concentration in the slow diffusion zone serves as a surrogate for the calcium current since it is directly related to the inward Ca 2+ driving force. The model of WT T-tubules containing a slow diffusion zone matches the experimental data (black curve – model, black circles – data). Removal of the diffusion barrier at the left side of the T-tubule in ( a ) results in a shorter initial delay as observed in the Bin1 HT experiments (red curve – model, red squares – data).

Article Snippet: To confirm the efficiency of Cre-mediated deletion of Bin1 , quantitative real-time PCR using TaqMan primer/probe sets (5'FAM/3'BHQ; Applied Biosystems) spanning constitutively splice exons 2 and 3 (Mm01158690_mH) was used to measure transcription of total Bin1 gene, the house-keeping gene Hrpt1 , and a cardiac specific gene TnI3 in cardiomyocyte cDNA generated from both WT and Bin1 HT mice.

Techniques: Diffusion-based Assay, Patch Clamp, Comparison, Concentration Assay

Journal: Nature medicine

Article Title: Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia

doi: 10.1038/nm.3543

Figure Lengend Snippet: Bin1 deletion increases extracellular K + diffusion, prolonging action potential duration and increasing ventricular ectopy. ( a ) Representative patch clamp recording of I K1 current changes when quickly switching extracellular potassium concentration in a wildtype (WT) cardiomyocyte. ( b ) Kinetics of I K1 during K + on in WT and Bin1 HT cardiomyocytes (dotted line, dead volume time of 124 ms). ( c ) Comparison of the initial delay X 0 of K + on for WT ( n = 20) and Bin1 HT ( n = 19) cardiomyocytes ( P = 0.0045). ( d ) Kinetics of I K1 during K + off (1−∆ I K1 ) in WT and Bin1 HT cardiomyocytes. ( e ) Comparison of X 0 of K + off for WT ( n = 20) and Bin1 HT ( n = 19) cardiomyocytes ( P = 0.0018). ( f ) Top: representative tracings of EKG (top) and TMP (transmembrane potential, bottom) from isolated and langendorff perfused WT (left) and Bin1 HT (right) hearts. Bottom: Action potential duration (APD80) is always prolonged in Bin1 HT hearts whether subjected to low (2.5 mM), normal (5 mM), and high (8 mM) potassium solution (left), and ventricular ectopy is increased in Bin1 HT hearts (right, incidence of arrhythmias during physiological buffer perfusion). ( g ) Ventricular activation map (left) and conduction velocity (right) of WT and Bin1 HT hearts subjected to high potassium (8 mM) perfusion (*, P < 0.05). Data are presented as mean ± SEM and cardiomyocytes are from three mice for each genotype, student’s t -test was used for statistical analysis.

Article Snippet: To confirm the efficiency of Cre-mediated deletion of Bin1 , quantitative real-time PCR using TaqMan primer/probe sets (5'FAM/3'BHQ; Applied Biosystems) spanning constitutively splice exons 2 and 3 (Mm01158690_mH) was used to measure transcription of total Bin1 gene, the house-keeping gene Hrpt1 , and a cardiac specific gene TnI3 in cardiomyocyte cDNA generated from both WT and Bin1 HT mice.

Techniques: Diffusion-based Assay, Patch Clamp, Concentration Assay, Comparison, Isolation, Activation Assay

Journal: Nature medicine

Article Title: Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia

doi: 10.1038/nm.3543

Figure Lengend Snippet: Ventricular arrhythmias induced by pacing and beta adrenergic activation with isoproterenol. ( a ) Representative recordings of EKG following a S1–S4 stimulation protocol. Normal sinus node beats resume immediately following pacing in WT mice (top panel), sustained monomorphic ventricular tachycardia (4.5 s) was induced in Bin1 HT mice (middle panel), sustained polymorphic ventricular tachycardia (VT) alternating with ventricular fibrillation (VF) (>20s) was induced in Bin1 HO mice (bottom panel). ( b ) Heart rate increase (∆HR) in response to isoproterenol was analyzed and compared among the three groups (mean ± SEM, n = 3–4, P = 0.04 by one-way ANOVA). ( c ) Incidence of sustained VT (>9 QRS) or VF in each group ( n = 3–4, P = 0.03 by chi-square). ( d ) The frequency of ventricular arrhythmias before and after isoproterenol treatment was quantified in each group ( n = 3–4, P < 0.01 by two-way ANOVA).

Article Snippet: To confirm the efficiency of Cre-mediated deletion of Bin1 , quantitative real-time PCR using TaqMan primer/probe sets (5'FAM/3'BHQ; Applied Biosystems) spanning constitutively splice exons 2 and 3 (Mm01158690_mH) was used to measure transcription of total Bin1 gene, the house-keeping gene Hrpt1 , and a cardiac specific gene TnI3 in cardiomyocyte cDNA generated from both WT and Bin1 HT mice.

Techniques: Activation Assay

Journal: Nature medicine

Article Title: Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia

doi: 10.1038/nm.3543

Figure Lengend Snippet: Adult mouse cardiomyocytes express four Bin1 splice variants. ( a ) Cartoon of Bin1 exons and the splice variants we found in adult mouse cardiomyocytes. BAR, Bin–Amphiphysin–Rvs domain; PI, phosphoinositide binding domain; CLAP, clathrin / AP2 binding region; MDB, myc-binding domain; SH3, SRC Homology 3 domain. ( b ) Four Bin1 splice variants with alternative inclusion of exon 13 and 17 are detected in adult mouse cardiomyocytes (A.M.C.) using PCR detection with primer sets flanking exon 10–18 or exon 13–18. ( c ) The percent of each Bin1 variants in adult mouse cardiomyocytes after subcloning and sequencing using PCR primer sets flanking exon 10–18. ( d ) Quantitative rtPCR analysis of each Bin1 variants ( Bin1/HPRT1 ) in purified neonatal cardiomyocytes (P3, n = 2 litters with 8–10 pups each) and isolated adult mouse cardiomyocytes ( n = 5 mice). ( e ) Western blot analysis confirms the antibody specificity of anti-exon 17 (clone 99D, Sigma) and anti-exon 13 (A#5299, Anaspec) BIN1 antibodies. All four BIN1 isoforms are detected by panBIN1 antibody (rabbit anti BIN1 SH3 domain). ( f ) Immunofluorescence of anti-exon 17 and anti-exon 13 labeling (red arrow, Z-line/TT region by α-actinin or Cav1.2 co-labeling) in adult mouse cardiomyocytes. ( g ) Representative confocal images (left, scale bars: 5 µm) and fluorescent profiles (right) of Di-8-ANNEPS membrane labeling in WT and Bin1 HT cardiomyocytes over-expressing GFP, BIN1, BIN1+13, BIN1+17, or BIN1+13+17 ( n = 5 cells). Data are presented as mean +/− SEM. *, P < 0.05; **, P < 0.01, and ***, P < 0.001 by student’s t -test or two-way ANOVA.

Article Snippet: To confirm the efficiency of Cre-mediated deletion of Bin1 , quantitative real-time PCR using TaqMan primer/probe sets (5'FAM/3'BHQ; Applied Biosystems) spanning constitutively splice exons 2 and 3 (Mm01158690_mH) was used to measure transcription of total Bin1 gene, the house-keeping gene Hrpt1 , and a cardiac specific gene TnI3 in cardiomyocyte cDNA generated from both WT and Bin1 HT mice.

Techniques: Binding Assay, Subcloning, Sequencing, Reverse Transcription Polymerase Chain Reaction, Purification, Isolation, Western Blot, Immunofluorescence, Labeling, Membrane, Expressing

Journal: Nature medicine

Article Title: Cardiac Spliced BIN1 Folds T-tubule Membrane, Controlling Ion Flux and Limiting Arrhythmia

doi: 10.1038/nm.3543

Figure Lengend Snippet: BIN1+13+17 uses F-actin to connect to Z-disc α-actinin. (a–b) HeLa cells expressing GFP tagged BIN1, BIN1+13, BIN1+17, and BIN1+13+17 (scale bars: 10 µm) ( a ), with the length of folds like structure (linear streaks) quantified in ( b ). (Mean ± SEM; n = 20 folds from 5 cells; *** indicates P < 0.001 by one-way ANOVA). ( c ) TEM confirms that BIN1+13+17 but not BIN1+17 induces elongated membrane folds in HeLa cells. Scale bars: 1 µm (left) and 0.5 µm (right two panels). ( d ) HeLa cells expressing isoforms of GFP-BIN1 (green) and LifeAct-mCherry (red) (scale bars: 10 µm). ( e ) GST pulldown of GST-BIN1 isoforms and N-WASP-V5 in HeLa cells. ( f ) In vitro pyrene-actin polymerization assay using purified Arp2/3, N-WASP and BIN1 isoforms. Left, representative tracing of actin polymerization kinetics. Right, the Vmax data of polymerization kinetics. Data are presented as mean ± SEM ( n = 5, * indicates P < 0.05 by one-way ANOVA). The negative control contains pyrene-actin alone with a GST control protein (GST-GFP, bottom black line indicated by the bottom arrow), the positive control contains pyrene-actin supplemented with Arp2/3 and VCA (active domain of N-WASP, top black line indicate by the top arrow), and the rest samples contain pyrene-actin supplemented with Arp2/3, N-WASP with GST-GFP or 1 µM GST-BIN1 isoforms. ( g ) Purified GST-BIN1 fusion protein pre-coated glutathione beads were added to adult heart lysates for pulldowns of α-actinin (right) or F-actin (left). ( h ) Schematic illustration of BIN1+13+17 forming an extracellular ionic diffusion barrier inside T-tubules.

Article Snippet: To confirm the efficiency of Cre-mediated deletion of Bin1 , quantitative real-time PCR using TaqMan primer/probe sets (5'FAM/3'BHQ; Applied Biosystems) spanning constitutively splice exons 2 and 3 (Mm01158690_mH) was used to measure transcription of total Bin1 gene, the house-keeping gene Hrpt1 , and a cardiac specific gene TnI3 in cardiomyocyte cDNA generated from both WT and Bin1 HT mice.

Techniques: Expressing, Membrane, In Vitro, Polymerization Assay, Purification, Negative Control, Control, Positive Control, Diffusion-based Assay

Journal: Frontiers in Immunology

Article Title: B7H3-dependent myeloid-derived suppressor cell recruitment and activation in pulmonary fibrosis

doi: 10.3389/fimmu.2022.901349

Figure Lengend Snippet: Clinical significance of circulating MDSCs and association with regulatory T cells and B7H3 + cells. Regression and correlation analyses were performed for MDSC abundance in peripheral blood and its relationship to lung function. The correlations between lung function (DLO, % predicted) and the frequencies of MDSCs (A) , G-MDSCs (B) , and M-MDSCs (C) are shown. N = 62. p < 0.05 in panels A–C. (D) The frequency of M-MDSCs in samples from the IPF patients without treatment was plotted for the correlation analysis. N = 17. p < 0.05. (E) The IPF patient samples were separated into untreated and treated (with pirfenidone or nintedanib) groups, and the M-MDSC frequency was compared between these two groups. N = 22 in untreated; N = 44 in treated IPF group. *p < 0.05. (F) Circulating CD4 + CD25 + regulatory T cells were analyzed in whole blood showing a positive correlation between the frequency of whole blood total MDSC and regulatory T cells. N = 31. p < 0.001. CD4 + CD25 + T-cell correlation between G-MDSC (G) and M-MDSC (H) is shown. N = 33. p < 0.001. The frequency of B7H3 + cells was analyzed, and the correlation with MDSC (I) , G-MDSC (J) , and M-MDSC (K) is shown. N = 42 in panels I and J, and 40 in panel (K) p < 0.05 in panel (I) , p < 0.01 in panel (J) , and p < 0.0001 in panel (K) MDSCs, myeloid-derived suppressor cells; DLCO, diffusing capacity of the lungs for carbon monoxide; G-MDSCs, granulocytic myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells.

Article Snippet: To assess the importance of B7H3 in vivo , blocking antibodies to mouse B7H3 (Bio X cell, Lebanon, NH, USA; Cat# BE0124; Clone# MJ18) or its isotype control rat IgG1 were injected into the mice i.v. via tail veins, every other day starting on day 1 after BLM treatment (0.3 mg/mouse).

Techniques: Derivative Assay

Journal: Frontiers in Immunology

Article Title: B7H3-dependent myeloid-derived suppressor cell recruitment and activation in pulmonary fibrosis

doi: 10.3389/fimmu.2022.901349

Figure Lengend Snippet: B7H3-activated MDSC promoted fibroblast activation/myofibroblast differentiation and suppressed T-cell proliferation. BM-derived MDSCs were treated with sB7H3 (4 µg/ml) or SCF (0.1 µg/ml) in presence of GM-CSF (0.01 µg/ml) for 3 days followed by flow cytometric cell sorting. Sorted G-MDSCs or M-MDSCs were co-cultured with primary isolated MLF in 24-well transwell plates (ratio of MDSC : MLF = 2:1). After 48 h of co-culture, RNA from MLF was isolated and analyzed by qPCR for type I collagen (Col1a2) (A) , α-SMA (Acta2) (B) , and TGFβ1 (Tgfb1) expression (C) . Cellular RNA from sorted G-MDSCs and M-MDSCs was also extracted and similarly analyzed for expression of TGFβ1 (Tgfb1) (D) . The 18S RNA was used as internal control for normalization. The data were expressed as fold change relative to the respective control. (E) B7H3 blocking antibody (Ab) was injected intravenously into mice after BLM treatment. The lung single-cell suspensions were obtained 7 days after BLM treatment for flow cytometry analysis of MDSCs. Representative plots (pre-gated by CD45 + CD11b + cells) are shown on the left panel and the quantitative analysis of percentages and absolute cell numbers on the middle and right panels, respectively. (F) Total lung cell numbers were counted using a hemocytometer. (G) Lung tissue RNA was analyzed by qPCR for Tnfa , Col1a1 , and Acta2 on day 7 after BLM treatment. (H) BM-derived CD11b + Gr1 + MDSCs with (+) or without (−) sB7H3 activation were co-cultured with CFSE pre-labeled splenocytes in media only (Naïve T cell) or in stimulation medium containing CD3/28 Dynabeads+rmIL2 (Activated T-cell) for 3 days. CD4 + or CD8 + T-cell proliferation was assessed by counting CFSE + cells with either T-cell marker using flow cytometry. (I) Fresh BM cells were treated with sB7H3 for 72 h and analyzed for CD84 and MDSC markers. The data were shown as the percentage (left) or the absolute numbers per million BM cells (right) of G-MDSC or M-MDSC in the CD84-expressing CD11b + BM population. Mean ± SD is shown for all. N = 3–8. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. MDSC, myeloid-derived suppressor cell; BM, bone marrow; sB7H3, soluble B7H3; SCF, stem cell factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MLF, mouse lung fibroblast; BLM, bleomycin; CFSE, carboxyfluorescein succinimidyl ester.

Article Snippet: To assess the importance of B7H3 in vivo , blocking antibodies to mouse B7H3 (Bio X cell, Lebanon, NH, USA; Cat# BE0124; Clone# MJ18) or its isotype control rat IgG1 were injected into the mice i.v. via tail veins, every other day starting on day 1 after BLM treatment (0.3 mg/mouse).

Techniques: Activation Assay, Derivative Assay, FACS, Cell Culture, Isolation, Co-Culture Assay, Expressing, Control, Blocking Assay, Injection, Flow Cytometry, Labeling, Marker

Journal: Frontiers in Immunology

Article Title: B7H3-dependent myeloid-derived suppressor cell recruitment and activation in pulmonary fibrosis

doi: 10.3389/fimmu.2022.901349

Figure Lengend Snippet: Lung scRNA-seq analysis identified B7H3-expressing cell clusters in human and mouse lung fibrosis. (A) Uniform manifold approximation and projection (UMAP) plots for all lung cells from IPF and control subjects were obtained from the IPF Cell Atlas online database ( CD276 expression was distributed in three distinct cell clusters shown in the top row). Diffusion map implementation labeled by cell type or disease status is shown in the middle row or the bottom row, respectively. (B) Level of CD276 in the lung cell clusters in IPF vs . control subjects. (C) Distribution of the expression signals for the indicated genes within the stromal cell cluster. (D) UMAP plots ( fibroXplorer.com ) of mouse lung fibroblasts identified as Dpt+ universal fibroblasts from BLM-treated lungs. Gene expression level for each gene is shown in the fibroblast sub-clusters. Lung fibroblast sub-clusters are color-coded (see left panel). (E) Pre-labeled MDSCs isolated from naïve BM were placed in the upper inserts. Cell-free CMs collected from untreated (control CM) or TGFβ-treated MLF (TGFβ-CM) cultures were incubated with B7H3 blocking antibody (Ab) or control IgG prior to placing in the lower chambers. The fluorescence intensity of the lower chamber was measured at 18 h of incubation. The data are presented as the percentages of TGFβ CMs over their respective control CMs. Mean ± SD are shown. N = 8/group. ****p < 0.0001. scRNA-seq, single-cell RNA sequencing; IPF, idiopathic pulmonary fibrosis; BLM, bleomycin; CMs, conditioned media; MLF, mouse lung fibroblast.

Article Snippet: To assess the importance of B7H3 in vivo , blocking antibodies to mouse B7H3 (Bio X cell, Lebanon, NH, USA; Cat# BE0124; Clone# MJ18) or its isotype control rat IgG1 were injected into the mice i.v. via tail veins, every other day starting on day 1 after BLM treatment (0.3 mg/mouse).

Techniques: Expressing, Control, Diffusion-based Assay, Labeling, Gene Expression, Isolation, Incubation, Blocking Assay, Fluorescence, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: B7H3-dependent myeloid-derived suppressor cell recruitment and activation in pulmonary fibrosis

doi: 10.3389/fimmu.2022.901349

Figure Lengend Snippet: Schematic illustration of the proposed model for B7H3-dependent MDSC role in pulmonary fibrosis. In response to signals from injured lung, hematopoietic progenitor cells (HPCs) proliferate and give rise to myeloid precursors (MPs) under the control of TERT, GM-CSF, and other potential factors. TERT-expressing MP may govern the origination/expansion of MDSCs at intermediate stage of myeloid cell differentiation. Two subtypes of MDSC (G- and M-MDSC) were expanded and activated with the stimulation of GM-CSF/sB7H3 produced by injured lung tissue. The sB7H3-recruited and/or activated MDSCs, in turn, were able to induce resident lung fibroblast activation and/or myofibroblast differentiation through TGFβ production in a paracrine manner and with the greater impact of M-MDSC, thus promoting lung fibrosis. Lung scRNA-seq analysis-identified macrophages and fibroblasts/myofibroblasts were potential cellular sources for induced B7H3 in injured lungs. The findings suggested the potential importance of the observed elevated circulating MDSCs in IPF pathogenesis. In addition, elevated CD84 + suppressive cell-enriched M-MDSC showed some correlation with Tregs in peripheral blood of IPF patients, suggesting MDSC facilitation of immunosuppressive cell network in IPF as well. MDSC, myeloid-derived suppressor cell; TERT, telomerase reverse transcriptase; GM-CSF, granulocyte-macrophage colony-stimulating factor; G-MDSCs, granulocytic myeloid-derived suppressor cells; M-MDSCs, monocytic myeloid-derived suppressor cells; IPF, idiopathic pulmonary fibrosis.

Article Snippet: To assess the importance of B7H3 in vivo , blocking antibodies to mouse B7H3 (Bio X cell, Lebanon, NH, USA; Cat# BE0124; Clone# MJ18) or its isotype control rat IgG1 were injected into the mice i.v. via tail veins, every other day starting on day 1 after BLM treatment (0.3 mg/mouse).

Techniques: Control, Expressing, Cell Differentiation, Produced, Activation Assay, Derivative Assay, Reverse Transcription

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A) Adult Na / Na . Feathers are absent on the neck and head, excepting the crown. (B) E8.5 embryos hybridized with a β - catenin probe to mark the patterning field and feather primordia. Punctate expression of β - catenin in feather placodes is seen on the body but not the neck of the mutant. WT, wild type; Na/Na , Naked neck. (C) E12.5 embryos showing limited lateral tract expansion (arrows) in Na / Na , reducing body feather coverage. (D) Quantitative RT-PCR determination of BMP12 expression in body and neck skin of E7.5 and E8.5 wild type and Na / Na embryos. (E,F) In situ hybridization detecting BMP12 in wild type and Na/Na embryos at (E) E7.5 and (F) E8.5. Wild type and mutant embryos were hybridized and photographed together. Na/Na embryos have elevated and diffuse expression of BMP12 in the skin. (G) Sequence traces of PCR products from E8.5 Na/+ . Genomic DNA PCR products display double peaks following a TA indel polymorphism in the BMP12 3′UTR. RT-PCR products from neck and body skin show a single trace throughout, indicating predominant expression of the Naked neck BMP12 allele, while both alleles are detected in RT-PCR products from other tissues. (H) Schematic showing insertion of chromosome 1 sequences into chromosome 3 at the Naked neck locus. Chromosome coordinates, the Naked neck identical by descent segment, gene names, exons, untranslated regions, and non-coding elements conserved between chicken and human genomes, based on the ENSEMBL genome viewer, are indicated.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, In Situ Hybridization, Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A) Application of recombinant BMP12 to cultured skin for 15 h leads to elevation of SOSTDC1 expression, determined by quantitative RT-PCR. (B–E) Detection of SOSTDC1 expression by in situ hybridization. (B) At E7.5 wild type embryos have two rows of feather placodes running up the neck. SOSTDC1 is expressed at the periphery of the placodes and is not detected in the medial region between the lateral rows of placodes. (C) By E8.5 the medial region of the neck is populated by feather placodes. (D) E7.5 Na/Na embryos have placodes on the dorsum, but widespread SOSTDC1 expression on the neck, including the medial region. (E) At E8.5 the Naked neck skin maintains a high level of widespread SOSTDC1 expression, with peri-placode expression visible on the body. (F) Ex vivo rescue of the Naked neck phenotype by suppression of BMP signaling. E7.0 Na/Na skin was cultured in the presence of dorsomorphin (DM, used at 8 µM) and SB203580 (SB, used at 5 µM), pharmacological inhibitors of BMP signal transduction, for 48 h. This permitted feather development across most of the mutant neck skin.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: Recombinant, Cell Culture, Expressing, Quantitative RT-PCR, In Situ Hybridization, Ex Vivo, Transduction, Mutagenesis

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A,B) β - catenin in situ hybridization revealing the effects of recombinant BMP12 application on feather periodicity and regional distribution in wild type skin after 48 h. (C,D) Dose effects of BMP12 on the number of feather placode rows on the spinal tract of the body. Feather primordia are visualized by β - catenin in situ hybridization. (E) SOSTDC1 expression on control and 80 ng/ml BMP12 treated skin explants. Feather placodes express SOSTDC1 at their periphery on both body and neck. Upon application of BMP12, the non-placode skin of the neck expresses a higher level of SOSTDC1 than does the body (compare signal intensity in the red boxed area to that of the blue boxed area). (F) Schematic of reaction-diffusion regulatory interactions. Adjacent numbering refers to mathematical terms in the supporting methods. C I represents the constitutive, ubiquitous production of the Inhibitor. (G) Quantification of periodicity of Activator foci in simulated neck and body with differential sensitivities to Inhibitor. C I increases along the x -axis. (H) Pattern outcomes from reaction-diffusion dynamics in a field with graded sensitivity to the Inhibitor. Abolition of Activator foci in the more sensitive part of the field is achieved with little effect on periodic spacing in the remainder of the field, producing a macropatttern that matches the effects of BMP12 treatment on cultured skin. Colors denote local Activator concentrations, with black representing the highest and white the lowest Activator levels. Areas with high Activator concentration represent placodes.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: In Situ Hybridization, Recombinant, Expressing, Control, Diffusion-based Assay, Cell Culture, Concentration Assay

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A) RA administration reduces the density of placodes, which are detected by β - catenin in situ hybridization, completely inhibiting placode formation at high doses. Suppression of BMP signaling with 4 µM dorsomorphin and 5 µM SB203580 rescues placode formation in the presence of RA. (B) Quantification of placode density on neck and body upon RA treatment. With increasing doses of RA the feather density on body and neck converges and ultimately all feather placode formation is suppressed. (C) RA sensitizes body skin to BMP-driven inhibition of feather development. The application of 0.1 µM RA has little effect on the placode pattern and application of 40 ng/ml BMP12 permits placode formation on the body. Co-treatment with RA and BMP12 has a synergistic effect, completely suppressing feather development on the body. Conversely, treatment of skin with the RA synthesis inhibitor Citral renders the neck resistant to suppression of placode formation by BMPs.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: In Situ Hybridization, Inhibition

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet: ( A ) Schematic representation of IRE1 with a C-terminal HaloTag, the construct used for tagging IRE1 at the endogenous locus. IF1 L and IF2 L refer to the primary dimerization and oligomerization interfaces of the lumenal domain, respectively. ( B ) RT-PCR analysis of stress-dependent XBP1 mRNA splicing in WT U-2 OS cells, IRE1 knock-out (KO) U-2 OS cells, and U-2 OS cells in which IRE1 has been fully edited with a C-terminal HaloTag. Tm indicates treatment with 5 μg/ml tunicamycin. ( C ) Immunoblot of UPR activation in response to 5 μg /ml tunicamycin (left) and 100 nM thapsigargin (right) treatments in the three cell lines shown in panel B. ( D ) Maximum intensity projections of representative spinning-disk confocal images of live cells expressing endogenously tagged IRE1-HaloTag, labeled with the JF549 dye. Regions shown with yellow boxes are enlarged below. ( E ) Same as D, except the cells have been treated with 5 μg/ml tunicamycin for 5 hr. Figure 1—source data 1. Annotated uncropped gel used to generate . Figure 1—source data 2. Raw uncropped gel used to generate . Figure 1—source data 3. All annotated uncropped gels used to generate . Figure 1—source data 4. Raw uncropped gel of immunoblot against IRE1 and phospho-IRE1 in . Figure 1—source data 5. Raw uncropped gel of immunoblot against XBP1 in . Figure 1—source data 6. Raw uncropped gel of immunoblot against PERK and actin in . Figure 1—source data 7. Raw uncropped gel of immunoblot against ATF4 in . Figure 1—source data 8. Raw uncropped gel of immunoblot against ATF6 in . Figure 1—source data 9. Raw uncropped gel of immunoblot against CHOP in .

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Western Blot, Activation Assay, Expressing, Labeling

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet: ( A ) Immunoblot showing IRE1 expression levels and UPR activation in WT U-2 OS cells, IRE1 KO U-2 OS cells, partial KO cells used as the parental cell line for generating HaloTag knock-ins, and two clones of endogenously labeled HaloTag (with high and low IRE1 expression levels). Note the shift in protein size due to the addition of the HaloTag and the absence of a WT IRE1 band in the two clones on the right. ( B ) Flow cytometry analysis of the low-and high-expressing clones shown in panel A. Cells were labeled with 5 nM JF549-HaloTag dye for 1 hr prior to the start of the flow cytometry experiment. Note the unimodal intensity distributions of both clones, ruling out the possibility that the lower-expressing clone simply contains a bimodal mixture of low- and high-expressing cells. Error bars represent 95% confidence intervals. Figure 1—figure supplement 1—source data 1. Annotated uncropped gel used to generate . Figure 1—figure supplement 1—source data 2. Raw uncropped gel of immunoblot against IRE1 and phospho-IRE1 in . Figure 1—figure supplement 1—source data 3. Raw uncropped gel of immunoblot against XBP1 in . Figure 1—figure supplement 1—source data 4. Raw uncropped gel of immunoblot against PERK in . Figure 1—figure supplement 1—source data 5. Raw uncropped gel of immunoblot against ATF4 and CHOP in . Figure 1—figure supplement 1—source data 6. Raw uncropped gel of immunoblot against ATF6 in . Figure 1—figure supplement 1—source data 7. Raw uncropped gel of immunoblot against actin in .

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Western Blot, Expressing, Activation Assay, Clone Assay, Labeling, Flow Cytometry

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet: Single-particle tracking data showing stress-dependent oligomerization of the high- and low-expressing IRE1-HaloTag clones. IRE1 in the lower-expressing clone remains dimeric in unstressed cells, while the shift to higher-order oligomers upon stress is less prominent than in the higher-expressing clone. Each data point represents a single cell. Error bars represent 95% confidence intervals. Figure 4—figure supplement 1—source data 1. Raw uncropped gel of immunoblot against IRE1, XBP1s, and GAPDH of . Figure 4—figure supplement 1—source data 2. Raw uncropped gel of immunoblot against PERK, ATF4, and CHOP of . Figure 4—figure supplement 1—source data 3. Raw uncropped gel of immunoblot against ATF6 of .

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Single-particle Tracking, Expressing, Clone Assay, Western Blot

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet:

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Cloning, CRISPR, Knock-Out, Expressing, Recombinant, Plasmid Preparation, Transfection, Sequencing, Software, Diffusion-based Assay, Single Particle

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Overexpression of AKAP95 in breast cancer tissues of 82 TNBC patient samples. From cBioPortal. Top, each box is a patient sample. Bottom, disease-free survival curves of patients with or without AKAP95 alterations. b, Growth assay for MDA-MB-231 cells expressing control or two AKAP95 shRNAs. Left, immunoblotting of total cell lysates and images of cell colonies stained with crystal violet. Right, numbers of cells in growth assays as mean ± SD from n = 3 independent experiments. c, Tumors from xenograft of control or AKAP95-KD MDA-MB-231 cells in immune-deficient mice. Tumor volumes at the indicated days post transplantation are plotted as mean ± SD (n = 9). d,e, RNA-seq analysis in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 and the indicated vector or AKAP95-expressing construct. d, Heatmap showing relative expression levels of genes down- or up-regulated in the indicated cells. It includes 951 and 294 genes down- and up-regulated in KD compared to control cells, respectively. Also see Supplementary Table 1, tab 1. e, GSEA for gene expression profiles of control and AKAP95-KD cells. Plots above and below the broken line show gene sets significantly enriched in up- and down-regulated genes by AKAP95 KD, respectively. f, Heatmap showing relative alternative splicing and clustered by changes in percent-spliced-in (PSI) values in the indicated cells. It includes 807 and 1275 alternative splicing events with decreased or increased PSI in KD cells, respectively. Also see Supplementary Table 1, tab 2. g, Gene ontology analysis for the indicated clusters from the heatmap in f. Blue (n = 807) and red (n = 1275) show functions significantly enriched in genes with PSI increase or decrease by AKAP95 KD, respectively. P values by log-rank test for a, Student’s t -test for b and d, and modified Fisher’s exact test for g. All two-sided. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Over Expression, Growth Assay, Expressing, Western Blot, Staining, Transplantation Assay, RNA Sequencing Assay, shRNA, Plasmid Preparation, Construct, Modification

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, CCNA2 expression in MDA-MB-231 cells upon AKAP95 KD. Left, relative mRNA levels of indicated cyclins were determined by RT-qPCR and normalized to GAPDH , and presented as mean ± SD from n = 3 biological repeats. Right, immunoblotting for Cyclin A1/A2 in total cell lysates. b, Co-overexpression of AKAP95 and CCNA2 in breast cancer tissues of 82 TNBC patients. Left, each box represents a patient. Right, correlation of their mRNA levels in the TNBC patients with indicated Pearson correlation coefficient. Both from cBioPortal. c,f, RNA immunoprecipitation-sequencing (RIP-seq) profiles for CCNA2 (c) and SMAD6 (f) based on our previous work . In blue are Anti-FLAG RIP-seq in control or 293 cells expressing the FLAG-HA-tagged AKAP95 WT or mutants. In red are anti-AKAP95 RIP-seq in control or AKAP95-KD 293 cells. In black are profiles of total input RNAs. All profiles have the same Y-axis scale. Red arrows indicate AKAP95-binding sites at intron 1. d, Total RNAs were used for RT-PCR, in the absence (-RT) or presence (+ RT) of reverse transcriptase, for CCNA2 intron 1 in MDA-MB-231 cells with indicated combination of siRNAs. Top, PCR products on agarose gel. Asterisk, an unknown amplification product. Repeated 3 times. Bottom, relative ratios of the signal for the intron 1-retaining transcript over the intron 1-spliced transcript, as mean ± SD from n = 3 independent experiments. e, Assay for CCNA2 mRNA stability. Control (Scr) and AKAP95-KD MDA-MB-231 cells were treated starting from 0 min with Actinomycin D (+A, to block RNA synthesis) and cycloheximide (+C, to block NMD) or not as indicated. Total RNA at indicated times were used for RT-PCR and normalized to ACTB , as mean ± SD from n = 3 biological repeats. g, mRNA-seq profiles for SMAD6 in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 (KD) and vector or AKAP95-expressing construct. Asterisk, a stop codon. P values by two-sided Student’s t -test for a and e and one-way ANOVA followed by Tukey’s post hoc test for d. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Immunoprecipitation, Sequencing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Blocking Assay, shRNA, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a-f, MDA-MB-231 cells were transduced with control or AKAP95 shRNA #1 (KD) and vector and FLAG-HA-tagged full-length AKAP95 WT or mutants. a, Immunoblotting of total cell lysates. Repeated 3 times. b, Colony formation assays. Left, colony numbers as mean ± SD from n = 3 biological repeats. Right, images of cells stained with crystal violet. c, Growth of cultured cells, as mean ± SD from n = 3 independent experiments. d, Relative SMAD6 mRNA level were determined by RT-qPCR and normalized to GAPDH , as mean ± SD from n = 3 biological repeats. e, RT-PCR for ratios for intron 1-retained over-spliced CCNA2 transcript, as mean ± SD from n = 4 biological repeats. f, RT-PCR for ratios for exon-included over-skipped RPUSD3 transcript, as mean ± SD from n = 3 biological repeats. g-k, MYC-transduced Akap95 KO MEFs were transduced with vector or constructs expressing HA-tagged full-length AKAP95 WT or mutants. g, Immunoblotting of total cell lysates. Repeated 3 times. h, Left, percentage of SA-beta-gal-positive cells as mean ± SD from n = 3 different images of MEFs from two embryos. Right, images from KO MEF 1. i, Relative mRNA levels of indicated genes, with related functions at bottom, were determined by RT-qPCR and normalized to Gapdh , as mean ± SD from n = 3 independent experiments. * or ** between vec and WT, WT and YS, WT and YF, except for Plk1, for which * only between vec and WT, WT and YF. *P<0.05, **P<0.01. j, Heatmap showing relative alternative splicing with PSI changes in MYC-transduced KO MEFs expressing indicated constructs (2 embryos each). Also see Supplementary Table 2, tab 5. k, Sashimi plot showing Aamdc alternative splicing that was rescued by introduction of AKAP95 WT, but not but the mutant, and RT-PCR for the inclusion of the alternative exon as mean ± SD from n = 4 biological replicates pooled from 2 embryos each. l, Diagram showing impact of material properties of AKAP95 WT and mutants on gene regulation and tumorigenesis. P values by two-sided Student’s t -test for c and one-way ANOVA followed by Tukey’s post hoc test for all other analyses. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transduction, shRNA, Plasmid Preparation, Western Blot, Staining, Cell Culture, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Construct, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a,b, MDA-MB-231 cells virally expressing control or indicated shRNAs ( a ) or shRNA combined with indicated constructs ( b ) were subject to immunoblotting of total cell lysates (top) and colony formation assay. Middle, colony numbers as mean ± SD from n = 3 ( a ) or 2 ( b ) independent experiments. Bottom, images of cells stained with crystal violet. c, Top, mRNA-seq profiles for CCNA2 in control or 293 cells of AKAP95 KD . The numbers of exon junction reads are indicated. The red asterisk at the gene diagram indicates a stop codon 57 bp downstream of exon 1 in the intron. The number of reads for the junction of exons 1 and 2, and for the average neighboring exons, and their ratios are in the tables below for indicated cells. d, Total RNAs were used for RT-PCR for intron 1 region in control and AKAP95-KD MDA-MB-231 cells treated with or without cycloheximide for 6 hours. Repeated > 3 times. e, Relative mRNA levels of UPF in UPF1-KD samples and BTZ in BTZ-KD samples, respectively, each relative to the control samples, as determined by RT-qPCR and normalized to GAPDH . f, Relative expression level of TGF-β pathway genes based on RNA-seq reads from control and AKAP95-KD MDA-MB-231 cells expressing vector or AKAP95. Venn diagram shows numbers of TGF-β pathway genes (from GSEA) downregulated by AKAP95 KD and upregulated by rescue with AKAP95 expression, and the relative expression of the 16 overlapped genes in both categories are plotted. g, RIP-seq profiles showing AKAP95 binding to RPUSD3 and PPM1K pre-mRNAs. Track information is the same as in . Red circles indicate the alternatively included exons (corresponding to the middle exon in the gene diagrams in ( h ), and red boxes show AKAP95 binding at the introns flanking these exons. h, Sashimi plots showing that the alternative splicing of RPUSD3 and PPM1K pre-mRNAs was affected by AKAP95 KD and rescued by restored expression of AKAP95. The numbers of exon junction reads and PSI are indicated. P values by two-sided Student’s t -test for a and b. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, shRNA, Construct, Western Blot, Colony Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, RNA Sequencing Assay, Plasmid Preparation, Binding Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a,b, MDA-MB-231 cells were virally infected to stably express scramble (control) or AKAP95 shRNA #1 (KD) and the indicated constructs including empty vector (vec) and FLAG-HA-tagged full-length AKAP95 WT or mutants. a, Relative CCNA2 mRNA level as determined by RT-qPCR and normalized to GAPDH , and plotted for each of the 2 biological repeats individually. b, RT-PCR for ratios for exon-included over-skipped PPM1K transcript, as mean ± SD from n = 3 biological repeats. c-f, MYC-transduced Akap95 KO MEFs were transduced with vector or constructs expressing HA-tagged full-length AKAP95 WT or mutants. c, Heatmap showing relative expression levels of genes changed in MYC-transduced KO MEFs (from 2 embryos each) stably expressing indicated rescue constructs. Also see Supplementary Table 2, tab 4. d, Relative mRNA levels of indicated SASP genes as determined by RNA-seq reads from n = 2 biological repeats (KO1 and KO2). e,f, Sashimi plots showing example genes for which the alternative exon inclusion was promoted ( e ) or suppressed ( f ) by introduction of AKAP95 WT, but not as effectively by YS or YF, and RT-PCR for the inclusion of the alternative exon, as mean ± SD from n = 2 embryos each. g, A model for how AKAP95 condensates may regulate gene expression for tumorigenesis. P values by one-way ANOVA followed by Tukey’s post hoc test. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Infection, Stable Transfection, shRNA, Construct, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transduction, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Growth of MEFs from Akap95 +/− (Het) and Akap95 −/− (KO) embryos (n = 6 each). b, Relative HRAS and MYC mRNA levels were determined by RT-qPCR and normalized to Actb , as mean ± SD from HRAS G12V and MYC transduced MEFs of 2 embryos each. c, HRAS-MYC-transduced MEFs in soft agar colony formation assay. Colony numbers as mean ± SD from n = 6 experiments using MEFs of 2 embryos each. d, Six mice received HRAS-MYC-transduced Het and KO MEFs on each flank. Tumor weights at four weeks are plotted. Each dot represents a tumor. e-i, MEFs derived from 3 KO and 3 Akap95-expressing (containing 1 WT and 2 Het) embryos were transduced with MYC. e, Right, images of cells before and after MYC transduction. Images of SA-beta-galactosidase activity assay are at the bottom. Relative MYC mRNA levels after transduction were determined by RT-qPCR and normalized to Actb (left top). Percentage of SA-beta-gal-positive cells are plotted (left bottom). Both as mean ± SD from MEFs (n = 3 embryos each). f, Heatmap showing relative expression levels of genes and clustered by changes in KO MEFs (2 embryos each). It includes 265 and 742 genes down- or up-regulated in KO, respectively. Also see Supplementary Table 2, tab 1. g, Gene ontology analysis for the indicated gene clusters from the heatmap in f. Blue (n = 265) and red (n = 742) show functions significantly enriched in down- and up-regulated genes, respectively. h, GSEA plots above and below the dashed line show gene sets significantly enriched in genes down- and up-regulated in the MYC-transduced KO compared to Het MEFs, respectively. i, Relative Akap95 and Ccna2 mRNA levels before and after MYC transduction as determined by RT-qPCR and normalized to Actb , as mean ± SD from MEFs from 3 KO and 2 WT/Het embryos. j, A diagram summarizing regulation of tumorigenesis by AKAP95 through gene expression control. P values by two-sided Student’s t -test for all except one-way ANOVA followed by Tukey’s post hoc test for i, and modified Fisher's exact test for g. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Quantitative RT-PCR, Soft Agar Assay, Derivative Assay, Expressing, Transduction, Activity Assay, Modification

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Immunoblotting for AKAP95 in HeLa cell nuclear extract and AKAP95 immunoprecipitation from the extract. Samples were boiled in the presence of DTT and resolved by SDS-PAGE. b, Disorder plot of human AKAP95. c, Turbidity by pictures and OD600 of MBP (none) and MBP fused to AKAP95 truncations at indicated concentrations all in 30 mM NaCl before and after TEV protease treatment for indicated time. OD600 is plotted as mean ± SD from n = 3 biological repeats. d, DIC (top) and fluorescence microscopy (bottom) images for 20 μM MBP-AKAP95 (101-210) and spiked with Oregon-green-labeled same protein (molar ratio 10:1) after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. e, Phase contrast images of 50 μM MBP-AKAP95 (101-210) in 30 mM NaCl in the absence and presence of 10% of PEG6000 after TEV protease treatment for 30 min. f, Fusion of two droplets formed by 50 μM MBP-AKAP95 (101-210) in 30 mM NaCl and 10% of PEG6000 after TEV protease treatment for 30 min. Also see Movie 1. g, DIC and fluorescence microscopy images of 6.25 μM MBP, MBP fused to Δ(101-210) or full-length AKAP95 in 150 mM NaCl, spiked with Oregon-green-labeled AKAP95 (101-210) at a molar ratio of 150:1 after TEV protease treatment for 30 min. Note that the lack of any condensates in the DIC images showed the inability of Δ(101-210) in condensation. Experiments in a, d, e-g were Repeated > 3 times. Scale bar, 5 μm for all. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Western Blot, Immunoprecipitation, SDS Page, Fluorescence, Microscopy, Labeling, Concentration Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, 293T cells were transfected with either empty vector (vec), or indicated AKAP95 construct with FLAG-HA-tag. Following α-Flag IP, the pulldown proteins were boiled and resolved by SDS-PAGE and detected by immunoblotting with α-HA. Blue and red asterisks indicate monomer and dimer, respectively. b, Identification of 1-100 as a probable prion subsequence on AKAP95. By the PLAAC program, using homo sapiens as background and core length of 30. c, Purified MBP and MBP fused to AKAP95 truncations as indicated or full-length AKAP95 (1-692) were resolved on SDS-PAGE and stained with Coomassie blue. d, MBP fused to AKAP95 truncations as indicated or full-length AKAP95 were resolved on SDS-PAGE and stained with Coomassie blue following treatment with TEV protease. Note that the cleaved MBP serves as a better indicator for cleavage efficiency as staining signal various for protein fragments of different sequences and sizes. e, Another event of fusion of two droplets formed by 50 μM MBP-AKAP95 (101-210) in 30 mM NaCl and 10% of PEG6000 after treatment with TEV protease for 30 min. Scale bar, 5 μm. Also see Movie S1. f, Quantification of nuclear AKAP95 concentration by anti-AKAP95 Western blot. Total lysates from indicated number of MDA-MB-231 (M231) and flp-TREx 293 cells (f293, un-induced and dox-induced for FH-AKAP95 expression) were loaded, along with indicated ng of purified MBP-AKAP95. AKAP95 signal of un-induced f293 is similar to that of 25 ng of MBP-AKAP95. All experiments were repeated 2 times. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transfection, Plasmid Preparation, Construct, SDS Page, Western Blot, Purification, Staining, Concentration Assay, Expressing

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Fluorescence microscopy images of Oregon-green-labeled AKAP95 (101-210) WT and YF at indicated protein and NaCl concentrations after TEV protease treatment for 30 min. Scale bar, 10 μm. Repeated > 3 times. b, Fluorescence microscopy images of 50 μM AKAP95 (101-210) WT and YF in 30 mM NaCl both spiked with Oregon-green-labeled (101-210) WT (molar ratio 150:1) after TEV protease treatment for 30’ and imaged immediately (30’) or after incubation for 60 (90’) or 120 (150’) more minutes. Scale bar, 10 μm. Repeated > 3 times. c, FRAP of 10 μM GFP-AKAP95 (101-210) WT and YF after 30 min of MBP cleavage in 150 mM NaCl and 10% of PEG6000. FRAP was performed immediately (30’) or after incubation for 60 min more (90’). Left, fluorescence microscopy images of droplets at indicated times. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery (relative to minimal level) at the final time. n = 7 droplets each. Scale bar, 2 μm. d, FRAP of Full-length AKAP95 WT and YF fused to GFP in HeLa cell nuclei. Left, fluorescence microscopy images of foci. The photobleached focus was boxed and amplified for the indicated time points. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery (relative to minimal level) at the final time. n = 7 cells each. Scale bar, 5 μm. e,f, Diffusion of full length AKAP95 WT and YF fused to GFP in HeLa cell nuclei, showing Line RICS normalized autocorrelation curves G(Ψ) as a function of the Spatial Lag (Ψ) ( e ) and diffusion coefficients ( f ), both as mean ± SD (n = 20 for WT, n = 22 for YF). g,h, Diffusion coefficients of purified GFP-AKAP95 (101-210) WT and YF, showing Line RICS normalized autocorrelation curves G(Ψ) ( g ) and diffusion coefficients ( h ), both as mean ± SD (n = 22 for WT, n = 13 for YF). P values by two-sided Student’s t -test (c, d) or Mann-Whitney U test (f, h). For box and whisker plots, data are median (line), 25–75th percentiles (box) and minimum-maximum values recorded (whiskers). Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Fluorescence, Microscopy, Labeling, Incubation, Amplification, Diffusion-based Assay, Purification, MANN-WHITNEY, Whisker Assay

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Immunostaining of endogenous AKAP95 (red) and DNA (DAPI, blue) in indicated cancer cell lines and primary MEFs from WT and Akap95 KO embryos. b, Confocal microscopy images of AKAP95 WT or ZF C-S fused to GFP in nuclei following transfection into HeLa cells. c, Fluorescence microscopy images of HeLa cells transiently expressing AKAP95 WT or Δ(101-210 fused to GFP. d, HeLa cells were transfected with AKAP95-GFP, and two nuclei were imaged at different time points. Time 0 was 24 hr after transfection. Note the growth and merge of the foci, especially those in the red circle. e, Rapid fusion of AKAP95 (ZF C-S )-GFP foci in a HeLa cell nucleus. The white oval and arrow show two different fusion events. These images are from movie 2. All experiments were Repeated > 3 times. Scale bar, 5 μm for all.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Immunostaining, Confocal Microscopy, Transfection, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1101/536839

Figure Lengend Snippet: a, Alignment of human and mouse AKAP95 (101-210). Middle row shows identical residues (by letter) and conservative mutations (by “+”). Tyr, red; Phe, blue and tall. Box, Tyr and Phe swapping. b, MBP alone (none) or MBP-AKAP95 (101-210) WT or mutants all at 50 μM and in 30 mM NaCl after TEV protease treatment for 30 min. Turbidity of each reaction was shown in pictures, and by OD600 as mean ± SD from n = 3 (for YA, YS) or 4 (the rest) independent assays. Samples taken after mixing (“mixed”) and from supernatant after centrifugation were resolved by SDS-PAGE followed by coomassie blue staining. c, DIC and fluorescence microscopy images for 10 μM Oregon-green-labeled MBP-AKAP95 (101-210) WT and mutants in 30 mM NaCl after TEV protease treatment for 30 min. Plots from left to right at bottom show the relative protein amount in droplet, number of droplets in a field, and the ratio of protein concentration inside droplets over sum of inside and outside droplets, respectively. Calculated as mean ± SD from n = 24 randomly picked droplets for WT or YF, except for number of droplets from n = 3 randomly picked fields. NA, not applicable. d, Fluorescence microscopy images of HeLa cells transfected with (top) and Flp-In T-Rex 293 cell lines expressing (bottom) GFP fusions with full-length AKAP95 WT or mutants. Repeated > 3 times. e,h, HEK293 cells co-transfected with indicated siRNAs and plasmids were subject to splice reporter assay (top) and immunoblotting with α-AKAP95 (bottom). Δ = Δ(101-210). Mean ± SD from n = 8 [except 5 for Δ(101-210) and 13 for YF and 2 nd WT] independent transfections are plotted in e and 7 independent transfections in h. f, Schematic of AKAP95 chimeras. g, Fluorescence microscopy images of 293T cells transfected with indicated AKAP95 chimeras fused to GFP. Repeated > 3 times. P values by two-sided Student’s t -test for b and one-way ANOVA followed by Tukey’s post hoc test for e and h. Scale bar, 5 μm for all. Uncropped blots are provided as source data.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301-062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Centrifugation, SDS Page, Staining, Fluorescence, Microscopy, Labeling, Protein Concentration, Transfection, Expressing, Reporter Assay, Western Blot

Journal: Brain

Article Title: Activated αβ T and reduced mucosa-associated invariant T cells in LGI1- and CASPR2-encephalitis

doi: 10.1093/brain/awaf096

Figure Lengend Snippet: Single-cell transcriptomics and flow cytometry identified expansion of plasma cells as a hallmark of LGI1-/CASPR2-AIE. ( A ) Sankey diagram showing overlap between LGI1-AIE, CASPR2-AIE, non-inflammatory-disease controls (IIH in cohort 1, functional disorder in cohort 2 and healthy controls in cohort 3) across the four sample cohorts. IHC = immunohistochemistry analysis of formalin-fixed, paraffin-embedded (FFPE) autopsy brain tissue. ( B ) Uniform manifold approximation and projection (UMAP) plot depicting the cell-type clusters of CSF cells. The second cell cluster included only PBMCs and is therefore shown only in . ( C – F ) Comparison of the relative cell-type abundance between LGI1 and IIH ( C ), CASPR2 and IIH ( D ), LGI1 and MS ( E ) and CASPR2 and MS ( F ). ( G ) Flow cytometry validation: relative percentage of plasma cells (%CD3 − CD19 + CD138 + ) quantified as percentages of all lymphocytes in CSF cells of the second cohort. Statistical significance was determined by the Kruskal–Wallis test with Dunn’s post hoc test and adjusted with the Benjamini–Hochberg method. FACS = fluorescence-activated cell sorting; FND = functional neurological disorders; IIH = idiopathic intracranial hypertension; MAIT = mucosal-associated invariant T cell; MS = multiple sclerosis.

Article Snippet: Homozygous MR1-deficient mice (MR1 AIE; LGI1 n = 3, CASPR2 n = 5) and C57BL/6 mice (C57BL6 AIE; LGI1 n = 2; CASPR2 n = 5) (10–15 weeks old, male and female) were immunized twice with recombinant mouse CNTNAP2 (>95% purity) or LGI1 protein (>90% purity) (Cntnap2-3316M, LGI1-9069M, Creative Biomart) emulsified in Complete Freund’s Adjuvant and supplemented with Mycobacterium tuberculosis H37Ra (4 mg/ml).

Techniques: Single-cell Transcriptomics, Flow Cytometry, Clinical Proteomics, Functional Assay, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Comparison, Biomarker Discovery, Fluorescence, FACS

Journal: Brain

Article Title: Activated αβ T and reduced mucosa-associated invariant T cells in LGI1- and CASPR2-encephalitis

doi: 10.1093/brain/awaf096

Figure Lengend Snippet: B-lineage cells in CSF are preferentially plasmablasts, clonally expanded, and express IgG1/2 and IgG4 heavy chains in LGI1-/CASPR2-AIE. Single-cell transcriptomes of all B-cell clusters (from ) in CSF and PBMCs from LGI1, CASPR2, IIH and MS patients were subclustered and analysed. ( A ) UMAP plot with B-lineage subclusters of CSF cells from AIE patients. Cluster 4 contains only PBMCs and is therefore not shown here. Expressions of selected marker genes are shown in and . Cluster 8 is composed of ‘contaminating’ T cells. ( B ) UMAP plot illustrating the distribution of B-cell clone sizes in CSF from AIE patients. UMAP depicting the immunoglobulin subtype transcribed by CSF cells from AIE patients. ( D ) Comparison of the frequency of transcribed immunoglobulin heavy chains (IGHG1 or IGHG2 versus IGHG4) in antibody-secreting B lymphocytes (ASCs) from CSF in all AIE patients. Only samples with >30 cells were considered. Significance was tested with a Mann–Whitney U-test. ( E and F ) Visual representation of B-cell receptor (BCR) clones (and clonotypes connected by lines) in the CSF (blue) and PBMCs (red) of two representative patients with LGI1-AIE ( E ) and CASPR2-AIE ( F ). Each dot indicates the amount of identical BCRs belonging to one clone. The area of each dot is proportional to the clone size. Clonotypes (connected by lines) were defined as identical VDJ gene segments, identical CDR3 length and a CDR3 nucleotide sequence with two or fewer mismatches. Asterisks mark clones that were verified experimentally as autoantigen specific. ( G ) Circos plot highlighting BCR clones composed of mixtures of plasmablasts, plasma cells and memory B cells. The line thickness indicates the number of cells. Clones consisting of only one cell type are marked in grey. AIE = autoimmune encephalitis; PBMC = peripheral blood mononuclear cell; UMAP = uniform manifold approximation and projection; VDJ = variable–diversity–joining.

Article Snippet: Homozygous MR1-deficient mice (MR1 AIE; LGI1 n = 3, CASPR2 n = 5) and C57BL/6 mice (C57BL6 AIE; LGI1 n = 2; CASPR2 n = 5) (10–15 weeks old, male and female) were immunized twice with recombinant mouse CNTNAP2 (>95% purity) or LGI1 protein (>90% purity) (Cntnap2-3316M, LGI1-9069M, Creative Biomart) emulsified in Complete Freund’s Adjuvant and supplemented with Mycobacterium tuberculosis H37Ra (4 mg/ml).

Techniques: Marker, Comparison, MANN-WHITNEY, Clone Assay, Sequencing, Clinical Proteomics

Journal: Brain

Article Title: Activated αβ T and reduced mucosa-associated invariant T cells in LGI1- and CASPR2-encephalitis

doi: 10.1093/brain/awaf096

Figure Lengend Snippet: T-cell repertoires in CSF of LGI1-/CASPR2-AIE patients show differential changes preferentially in the CD4 TCM clusters, clonal expansion of activated CD4 TCM and CD8 TCM clusters, and blood–CSF-spanning T-cell clones. ( A ) UMAP plot based on the T-cell subclustering (of all T-cell clusters from ) of all PBMCs and CSF cells. ( B and C ) Unbiased, cluster-free analysis of differential T-cell abundance was analysed in a pairwise fashion using the DAseq tool and visualized in shades of red for increases and shades of blue for decreases; comparison of LGI1 and IIH CSF T cells ( B ) and CASPR2 and IIH CSF T cells ( C ). ( D ) T-cell clone sizes in CSF from AIE patients projected onto the UMAP plot. ( E and F ) Visual representation of T-cell receptor clones in CSF (blue) and PBMC (red) of two representative patients with LGI1-AIE ( E ) and CASPR2-AIE ( F ). In the network, each dot indicates one clone (identical VDJ genes and CDR3 regions). The area is proportional to the clone size. The pie charts indicate the percentage of CSF cells and PBMCs within each clone. AIE = autoimmune encephalitis; IIH = idiopathic intracranial hypertension; MAIT = mucosal-associated invariant T cell; NK = natural killer cell; PBMC = peripheral blood mononuclear cell; TCM = central memory T cell; UMAP = uniform manifold approximation and projection; VDJ = variable–diversity–joining.

Article Snippet: Homozygous MR1-deficient mice (MR1 AIE; LGI1 n = 3, CASPR2 n = 5) and C57BL/6 mice (C57BL6 AIE; LGI1 n = 2; CASPR2 n = 5) (10–15 weeks old, male and female) were immunized twice with recombinant mouse CNTNAP2 (>95% purity) or LGI1 protein (>90% purity) (Cntnap2-3316M, LGI1-9069M, Creative Biomart) emulsified in Complete Freund’s Adjuvant and supplemented with Mycobacterium tuberculosis H37Ra (4 mg/ml).

Techniques: Clone Assay, Comparison

Journal: Brain

Article Title: Activated αβ T and reduced mucosa-associated invariant T cells in LGI1- and CASPR2-encephalitis

doi: 10.1093/brain/awaf096

Figure Lengend Snippet: Patients with LGI1-/CASPR2-AIE show trans-compartment loss of innate-like MAIT cells and shifts in the NK cell subpopulations. ( A ) UMAP showing the subclustering of the mixed T-cell cluster (cluster 10, ). The expression levels of marker genes for each cell type shown are visualized in . ( B ) Heat map comparing selected cell-type abundances (of the subclustering shown in ) between AIE patients, IIH and MS controls across CSF and PBMC compartments. Colours indicate an increase (red) or decrease (blue), and asterisks indicate significance ( and ). ( – E ) Flow cytometry validation: cell proportions of dnTc (CD3 + CD4 − CD8 − ) , CD56dim NK cells (CD3 − CD56 dim ) ( D ) CD56bright NK cells ( E ), in the CSF (cohort 2). ( F ) Cell proportion of MAIT cells in PBMCs in another independent flow cytometry cohort (cohort 3). Frequency respective to parent gate. Gating schemes are shown in (cohort 2) and (cohort 3). AIE = autoimmune encephalitis; FND = functional neurological disorders; IIH = idiopathic intracranial hypertension; MAIT = mucosal-associated invariant T cell; MS = multiple sclerosis; NK = natural killer cell; PBMC = peripheral blood mononuclear cell.

Article Snippet: Homozygous MR1-deficient mice (MR1 AIE; LGI1 n = 3, CASPR2 n = 5) and C57BL/6 mice (C57BL6 AIE; LGI1 n = 2; CASPR2 n = 5) (10–15 weeks old, male and female) were immunized twice with recombinant mouse CNTNAP2 (>95% purity) or LGI1 protein (>90% purity) (Cntnap2-3316M, LGI1-9069M, Creative Biomart) emulsified in Complete Freund’s Adjuvant and supplemented with Mycobacterium tuberculosis H37Ra (4 mg/ml).

Techniques: Expressing, Marker, Flow Cytometry, Biomarker Discovery, Functional Assay

Journal: Brain

Article Title: Activated αβ T and reduced mucosa-associated invariant T cells in LGI1- and CASPR2-encephalitis

doi: 10.1093/brain/awaf096

Figure Lengend Snippet: MAIT cells are present in the brain of patients with CASPR2-AIE and LGI1-AIE, and their absence leads to reduced presence of autoantibodies in a murine immunization model. ( A ) Multiplexed immunofluorescence (mIF) staining of post-mortem CASPR2-AIE brain tissue, showing CD3 (red), CD8 (green), DAPI (white), CD4 (yellow) and CD161 (blue). Scale bars = 1 mm in overview; 20 μm in subpanels. ( B and C ) Box plots representing CD3 + CD161 + CD4-gdTc-CD8 +/− T cells as a percentage of CD3 + cells in the parenchyma ( B ) and meninges ( C ) of three human CASPR2- and three LGI1-AIE patients as shown in multiplex immunohistochemistry. ( D and E ) Murine immunization model using full-length LGI1/CASPR2 protein immunization (AIE) versus sham immunizations (control) of MAIT-deficient mice (MR1) and littermates (C57BL6). Serum was analysed on Day 28 for LGI1/CASPR2 antibody positivity using a cell-based assay, and the end point titre was determined. ( D ) Bar plot depicting percentage of antibody-positive mice. ( E ) Box plot showing antibody titre with respect to their disease group. Statistical significance was determined by Fisher’s exact test in D and by Wilcoxon rank test in E . AIE = autoimmune encephalitis; IHC = immunohistochemistry; MAIT = mucosal-associated invariant T cell.

Article Snippet: Homozygous MR1-deficient mice (MR1 AIE; LGI1 n = 3, CASPR2 n = 5) and C57BL/6 mice (C57BL6 AIE; LGI1 n = 2; CASPR2 n = 5) (10–15 weeks old, male and female) were immunized twice with recombinant mouse CNTNAP2 (>95% purity) or LGI1 protein (>90% purity) (Cntnap2-3316M, LGI1-9069M, Creative Biomart) emulsified in Complete Freund’s Adjuvant and supplemented with Mycobacterium tuberculosis H37Ra (4 mg/ml).

Techniques: Immunofluorescence, Staining, Multiplex Assay, Immunohistochemistry, Control, Cell Based Assay

Journal: Nucleic Acids Research

Article Title: Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation

doi: 10.1093/nar/gkw844

Figure Lengend Snippet: ( A ) Using peak calling on two replicates, 9213 STAT5A enhancers were found to coincide with H3K27ac (±500 bp) at day one of lactation (L1). A total of 4372 of them decommissioned within 12 h upon termination of lactation (class 1), 4526 within 24 h (class 2) and 315 were resilient (class 3). Since termination of lactation coincides with the onset of involution, these two time points were labeled I12 and I24. ( B ) Peak profile of STAT5A and H3K27ac based on the 9213 STAT5A peaks and STAT5A ChIP-seq data from wild-type tissue at L1 (blue), I12 (dark green) and I24 (light green). The coverage at I12 was reduced by 15% and by 90% at I24. H3K27ac ChIP-seq data from wild-type tissue at L1 (blue), I12 (dark green) and I24 (light green) are shown. The reduction in acetylation was larger at I12 than at I24. ( C ) A representative gene is shown for each of the enhancers groups. The left panel illustrates Glycam1 , a class 1 gene, with ‘very sensitive’ enhancers. The four enhancers completely decommissioned within 12 h, as did H3K27ac. The middle panel shows Olah , a ‘less sensitive’ class 2 enhancer. Enhancer height decreased at I12, but all of them were still present. Also acetylation had decreased at I12. However, all STAT5A enhancers as well the acetylation were non-existent after 24 h. The right panel shows Bcl6 , a class 3 gene with ‘resilient enhancers’, which were not affected by involution. The STAT5A enhancers as well as the acetylation remained stable throughout involution. ( D ) STAT5 enhancers within 10 kb of the nearest TSS of an annotated gene. By selecting enhancers within 10 kb of TSS, a region associated with enhancers , a total of 1191 class 1 enhancers, 1304 class 2 enhancers and 61 class 3 enhancers were identified. ( E ) The bar plot illustrates that the most stable enhancers were more likely associated with GAS motifs, as only 38% of the class 1 have a GAS motif within 200 bp. This percentage increased to 54% for the 1304 class 2 enhancers and 74% for class 3 enhancers, which were still present at I24. ( F ) Coverage of class 1 enhancers was higher in the presence of GAS motifs than without. ( G ) Enhancer height increased in class 2 enhancers, but the coverage of enhancers with an underlying GAS motif was still higher. ( H ) Class 3 enhancers displayed the highest coverage and the ones with GAS motif were again higher than those without. ( I ) Peak coverage at L1 of the three classes, independent of an underlying GAS motif. The resilient class 3 enhancers had the highest coverage, followed by class 2 and class 1 enhancers. Color code in panels A, D-I, class 1 (purple), class 2 (brown) and class 3 (yellow).

Article Snippet: Complementary DNA was synthesized from total RNA using SuperScript II (Invitrogen) and quantitative PCR was performed using the Taqman probe-based system ( Stat5a , Mm00839861_m1; mouse Gapdh endogenous control, 4352339E, Applied Biosystem) on the CFX384 Real-Time PCR Detection System (Bio-Rad).

Techniques: Labeling, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation

doi: 10.1093/nar/gkw844

Figure Lengend Snippet: ( A ) Co-binding of STAT5A and GR. Fifty-one percent of the STAT5A enhancers coincided with GR and out of those 43% (562) were classified as class 1 enhancers (30% contain GAS motif), 53% (691) as class 2 (51% contain GAS motif) and 4% (45) as class 3 enhancers (67% contain GAS motif). ( B ) Temporal decommission of GR. The peak coverage was reduced by 35% within 12 h, and by 91% within 24 h, equivalent to that observed for STAT5. ( C ) Genes representing the three differential STAT5 enhancers. Wap (class 1), with its three enhancers, showed the same decommission pattern for STAT5A and GR. The most distal enhancers (E3) was lost within 12 h (I12) and no enhancers remained at 24 h (I24) of involution. All enhancers in Olah (class 2) were intact at I12 but completely decommissioned at I24. However, the enhancer height for STAT5 and GR was already reduced at I12. Bcl6 (class 3) showed no GR co-binding compared to the classes 1 and 2. ( D ) Coverage plots for STAT5A and GR enhancers in each of the enhancer categories. STAT5A and GR enhancer coverage was equivalent at class 1 sites. At class 2 sites GR coverage was smaller than that of STAT5A and class 3 sites had relatively the smallest GR enhancers.

Article Snippet: Complementary DNA was synthesized from total RNA using SuperScript II (Invitrogen) and quantitative PCR was performed using the Taqman probe-based system ( Stat5a , Mm00839861_m1; mouse Gapdh endogenous control, 4352339E, Applied Biosystem) on the CFX384 Real-Time PCR Detection System (Bio-Rad).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation

doi: 10.1093/nar/gkw844

Figure Lengend Snippet: ( A ) Class 1 STAT5A enhancers were separated into enhancers with and without GAS motif. Thirty-eight percent of the enhancers with, and 53% without, a GAS motif co-bound GR. ( B ) STAT5 enhancers were higher in the presence of GAS motifs. Coverage of GR was independent of the GAS motif status. ( C ) Preferential reduction of class 1 enhancers containing a GAS motif occurred at 12 h of involution (I12). GR coverage at enhancers with GAS motifs is less than 45% of that obtained at enhancers lacking a GAS motif. At sites without GAS motif GR enhancers continue to exceed STAT5A enhancers. ( D ) Peak coverage at 24 h of involution (I24). At this time point most enhancers have been decommissioned but the pattern is reminiscent to that seen at I12. ( E ) Peak coverage of STAT5A class 1 enhancers with and without GAS motifs and GR, ELF5 and NFIB co-binding. Enhancers without GAS motif showed stronger co-binding of GR, ELF5 and NFIB as compared to those with a GAS motif. ( F ) Three representative genes and their STAT5A enhancers illustrate co-binding. Glycam1 , GR co-bound at all STAT5A enhancers. Strongest ELF5 binding was at the enhancer lacking a GAS motif (asterix). The STAT5A enhancer without GAS motif at Olah showed the strongest ELF5 and NFIB co-binding. All STAT5A enhancers co-bound GR. Csn2 had also one STAT5A enhancer without GAS motif, which had the strongest co-binding by GR and ELF5.

Article Snippet: Complementary DNA was synthesized from total RNA using SuperScript II (Invitrogen) and quantitative PCR was performed using the Taqman probe-based system ( Stat5a , Mm00839861_m1; mouse Gapdh endogenous control, 4352339E, Applied Biosystem) on the CFX384 Real-Time PCR Detection System (Bio-Rad).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation

doi: 10.1093/nar/gkw844

Figure Lengend Snippet: ( A ) Alveolar development is dependent on the Stat5 autoregulatory enhancer. Histologically mammary tissue appeared normal in the presence of only one intact Stat5 allele ( Stat5 +/− ). Mammary tissue with homozygous deletion of the autoregulatory enhancer ( Stat5 ΔE/ΔE ) appeared less differentiated and Stat5 ΔE/− was severely underdeveloped. ( B ) Qualitatively, pSTAT5 staining of wild-type ( Stat5 +/+ ) tissue appeared similar to Stat5 +/− . In contrast, pSTAT5 staining was greatly reduced in Stat5 ΔE/ΔE , Stat5 ΔE/− tissues. ( C ) Stat5a mRNA levels in wild-type and mutant mammary tissue ( Stat5 +/+ n = 6; Stat5 +/- n = 2; Stat5 ΔE/ΔE n = 4; Stat5 ΔE/− n = 3). ( D ) The Western blot showed reduced STAT5 levels in Stat5 ΔE/ΔE tissue. GAPDH served as control, and showed no reduction. ( E ) Peak coverage in wild-type and mutant tissue. ( F ) STAT5A enhancers were reduced in Stat5 +/− tissue and completely absent in Stat5 ΔE/ΔE and Stat5 ΔE/− tissue. Reduction of H3K27ac was delayed in all genotypes except Stat5 ΔE/− where it was absent. ( G ) Representative genes with class 1, class 2 and class 3 enhancers. STAT5 binding in mutant tissue was most severely affected in class 1 enhancers ( Glycam1 ) and the least in class 3 enhancers ( Bcl6 ).

Article Snippet: Complementary DNA was synthesized from total RNA using SuperScript II (Invitrogen) and quantitative PCR was performed using the Taqman probe-based system ( Stat5a , Mm00839861_m1; mouse Gapdh endogenous control, 4352339E, Applied Biosystem) on the CFX384 Real-Time PCR Detection System (Bio-Rad).

Techniques: Staining, Mutagenesis, Western Blot, Control, Binding Assay

Journal: Nucleic Acids Research

Article Title: Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation

doi: 10.1093/nar/gkw844

Figure Lengend Snippet: ( A ) Decline of active STAT5 in wild-type and mutant mammary tissue. Phospho-STAT5-positive cells in wild-type tissue decreased within 12 h (I12) after terminating lactation and were absent at 24 h (I24). In contrast no pSTAT5-positive cells were observed at I12 in tissue lacking the autoregulatory enhancer Stat5 ΔE/ΔE . ( B ) A total of 5255 out of 9213 STAT5A binding sites (enhancers) in wild-type tissue were shared with Stat5 ΔE/ΔE tissue at L1, suggesting that the full establishment of enhancer was not accomplished at lower STAT5 levels. ( C ) The coverage plot illustrates that the wild-type L1 sample had the highest coverage. Even wild-type tissue at I12 showed a higher coverage than the Stat5 ΔE/ΔE samples at L1. Stat5 ΔE/ΔE at involution 12 h showed the lowest coverage. ( D ) Heat map comparing STAT5A coverage in wild-type and Stat5 ΔE/ΔE tissue in the three different enhancer categories. ( E ) Representative examples from the heat map. The STAT5A enhancer in the Stat5a locus was disrupted in Stat5 ΔE/ΔE tissue. H3K27ac coverage was reduced but still present. STAT5 binding to the Wap enhancers was greatly reduced in Stat5 ΔE/ΔE tissue. Class 3 enhancers were the least affected in mutant tissue. The height of the STAT5A enhancers was lower in the Stat5 ΔE/ΔE sample, but H3K27ac remained unaltered.

Article Snippet: Complementary DNA was synthesized from total RNA using SuperScript II (Invitrogen) and quantitative PCR was performed using the Taqman probe-based system ( Stat5a , Mm00839861_m1; mouse Gapdh endogenous control, 4352339E, Applied Biosystem) on the CFX384 Real-Time PCR Detection System (Bio-Rad).

Techniques: Mutagenesis, Binding Assay

Journal: Nucleic Acids Research

Article Title: Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation

doi: 10.1093/nar/gkw844

Figure Lengend Snippet: ( A ) Homeostasis between STAT5 and STAT3. While pSTAT5 levels declined upon termination of lactation, pSTAT3 levels increased. ( B ) Binding sites shared by STAT5 and STAT3 were identified by integrating STAT5A data at L1 and STAT3 data at I24. ( C ) Bar plot showing the reduction of STAT5A binding compared to the increase of STAT3 binding during involution. This indicates that STAT3 replaced STAT5, but not necessarily at the same binding sites. The venn diagrams illustrate the overlap of STAT5A and STAT3 enhancers for L1, I12 and I24. Due to the replacement of those two factors most overlaps could be detected at involution 12 h. ( D ) STAT3 binding to the three enhancer categories. Six percent (280) of class 1 STAT5A sites overlapped with STAT3. Thereby, STAT3 and H3K27ac peak height was reduced from L1 to I12, but not from I12 to I24. Twenty-five percent (1137) of class 2 enhancers co-bound STAT3, but by looking at the peak coverage during involution the H3K27ac decreased and STAT3 increased. The last group of common enhancers had 173 enhancers (55%) overlapping STAT5A and STAT3 enhancers and the coverage plot shows that the peak coverage of STAT3 increased and H3K27ac decreased slightly during involution. ( E ) Representative genes to demonstrate the STAT5/3 homeostasis. Glycam1 (class 1) displayed STAT3 co-binding at the promoter at L1, but no increase of STAT3 at the individual enhancers was observed. Wap (class 1) had no STAT3 co-binding at the third enhancer (E3). However, E1 and E2, which decommissioned at I24, co-bound STAT3 at I12 and even stronger at I24. The promoter showed continuous co-binding. The class 2 representative, Lalba , displayed constant co-binding at the promoter, and the enhancers showed a co-binding starting at involution 12 h, getting stronger at I24, when the STAT5A enhancers were absent. The resilient Bcl6 gene (class 3) showed continuous co-binding during involution.

Article Snippet: Complementary DNA was synthesized from total RNA using SuperScript II (Invitrogen) and quantitative PCR was performed using the Taqman probe-based system ( Stat5a , Mm00839861_m1; mouse Gapdh endogenous control, 4352339E, Applied Biosystem) on the CFX384 Real-Time PCR Detection System (Bio-Rad).

Techniques: Binding Assay