Journal: The Journal of Physiology

Article Title: Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca 2+ influx through cyclic nucleotide‐gated channels

doi: 10.1113/JP273150

Figure Lengend Snippet: A, the increase in the dye diffusion distance induced by 2‐PAA (20 μm, 1 h) was not blocked by the protein kinase A inhibitors Rp‐cAMPS (Rp, 200 μm) and KT5720 (KT, 1 μm). Additionally, the activator of the exchange protein directly activated by cAMP, 8‐pCPT‐O‐Me‐cAMP (100 μm) did not affect the dye diffusion distance. B, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem (L‐cis‐dil., 100 μm) could prevent the increase in the dye diffusion distance induced by 2‐PAA (20 μm, 1 h) relative to the vehicle control (cont., 0.3% ethanol) in scrape loading/dye transfer assays. C, RT‐PCR showed that the CNG channel subunits A1, A2 and B1 were expressed in hCMEC/D3 cells. D, furthermore, the cAMP‐sensitive subunit CNGA2 was confirmed to be expressed at protein level with β‐tubulin (β‐Tb) serving as loading control. E, in whole‐cell patch‐clamp experiments, 2‐PAA (20 μm) increased the current measured in hCMEC/D3 cells. This increased current was completely abolished by simultaneous application of the CNG channel blocker l‐cis‐diltiazem (L‐cis‐dil., 100 μm) with 2‐PAA. All results were analysed using Student's t test. *Significant differences to the vehicle control: * P < 0.05, ** P < 0.01, *** P < 0.001; #significant differences to 2‐PAA: # P < 0.05.

Article Snippet: Anti‐β‐tubulin antibody for the loading control (Sigma‐Aldrich, T4026) was diluted 1:7500, anti‐CNGA2 antibody (Alomone Labs, Jerusalem, Israel, APC‐045) was diluted 1:750 and anti‐Cx37 antibody (Abcam, ab58918) was diluted 1:700 in TBS‐T and applied to the membranes at 4 °C overnight.

Techniques: Diffusion-based Assay, Reverse Transcription Polymerase Chain Reaction, Patch Clamp

Journal: The Journal of Physiology

Article Title: Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca 2+ influx through cyclic nucleotide‐gated channels

doi: 10.1113/JP273150

Figure Lengend Snippet: List of all primer pairs used for gene expression analyses and quantitative real time PCR

Article Snippet: Anti‐β‐tubulin antibody for the loading control (Sigma‐Aldrich, T4026) was diluted 1:7500, anti‐CNGA2 antibody (Alomone Labs, Jerusalem, Israel, APC‐045) was diluted 1:750 and anti‐Cx37 antibody (Abcam, ab58918) was diluted 1:700 in TBS‐T and applied to the membranes at 4 °C overnight.

Techniques: Expressing, Sequencing, Amplification

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: bioRxiv

Article Title: Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

doi: 10.1101/2024.02.28.582540

Figure Lengend Snippet: Nanopore sequencing results of an AIV viral culture using DNA-nanopores (“cDNA” sequencing through R10 chemistry; direct RNA sequencing through “RNA002” R9 chemistry) and RNA-nanopores (direct RNA sequencing through “RNA004” RNA chemistry). A. Sequencing read length distribution across the cDNA, RNA002, and RNA004 datasets. B. Reference genome coverage of the three sequencing datasets across all AIV segments (PB1: Polymerase basic 1, PB2: Polymerase basic 2, PA: Polymerase acidic, HA: Hemagglutinin, NP: Nucleoprotein, NA: Neuraminidase, M: Matrix, NS: Nonstructural). The horizontal line indicates a coverage of 50x.

Article Snippet: Here we find that the latest direct RNA nanopore sequencing technology (which is based on a unique RNA-nanopore specifically designed for transcriptomic rather than genomic research), provides similar results to cDNA sequencing using Oxford Nanopore Technologies’ established high-accuracy DNA-nanopores (R10 chemistry).

Techniques: Nanopore Sequencing, Sequencing, RNA Sequencing

Journal: bioRxiv

Article Title: Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

doi: 10.1101/2024.02.28.582540

Figure Lengend Snippet: Evaluation of viral consensus sequence creation from nanopore sequencing datasets (cDNA, RNA002, RNA004) across all data rarefactions ( min , med , max ). The performance of the computational tools BCFtools, iVar, and IRMA, which were the best-performing approaches for the max datasets, is visualized. A. Consensus sequence evaluation across the eight viral AIV segments through normalized BIT scores calculated based on the known AIV reference. B. Consensus sequence evaluation through whole-genome evolutionary distance comparisons with the known AIV reference.

Article Snippet: Here we find that the latest direct RNA nanopore sequencing technology (which is based on a unique RNA-nanopore specifically designed for transcriptomic rather than genomic research), provides similar results to cDNA sequencing using Oxford Nanopore Technologies’ established high-accuracy DNA-nanopores (R10 chemistry).

Techniques: Sequencing, Nanopore Sequencing

Journal: bioRxiv

Article Title: Latest RNA and DNA nanopore sequencing allows for rapid avian influenza profiling

doi: 10.1101/2024.02.28.582540

Figure Lengend Snippet: Phylogenetic tree of AIV consensus HA segments from four environmental samples (dust samples from turkey farm in France) and known European AIV strains. AIV of the environmental samples was assessed by cDNA nanopore sequencing, and consensus sequence was created through BCFtools.

Article Snippet: Here we find that the latest direct RNA nanopore sequencing technology (which is based on a unique RNA-nanopore specifically designed for transcriptomic rather than genomic research), provides similar results to cDNA sequencing using Oxford Nanopore Technologies’ established high-accuracy DNA-nanopores (R10 chemistry).

Techniques: Environmental Sampling, Nanopore Sequencing, 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: 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: 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