Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Ectopic Fatty Acid–Binding Protein 4 Expression in the Vascular Endothelium is Involved in Neointima Formation After Vascular Injury

doi: 10.1161/JAHA.117.006377

Figure Lengend Snippet: Effects of the conditioned medium of Fabp4 ‐overexpressed endothelial cells in vascular smooth muscle cells. A, Experimental design of human coronary artery smooth muscle cells ( HCASMCs ) treated with the conditioned medium ( CM ) prepared by 24‐hour incubation of adenovirus vector of fatty acid–binding protein 4 (Ad‐ FABP 4)– and empty sequence (Ad‐Control)–transfected human coronary artery endothelial cells ( HCAECs ) in the absence and presence of 10 μg/mL anti‐ FABP 4 antibody ( FABP 4‐Ab) for 24 hours ( HCASMC ‐ CM ‐Ab). B, Gene expression of inflammatory cytokines and proliferation‐ and adhesion‐related molecules determined by quantitative real‐time polymerase chain reaction (PCR) in HCASMC ‐ CM ‐Ab (n=6 in each group). * P <0.05 vs CM of Ad‐Control–transfected HCAECs ( CM ‐Ad‐Control). † P <0.05 vs CM of Ad‐ FABP 4–transfected HCAECs ( CM ‐Ad‐ FABP 4). C and D, Proliferation of HCASMC ‐ CM supplemented with 5% FBS in the absence and presence of 10 μg/mL FABP 4‐Ab for 24 hours assessed by MTS (C) and bromodeoxyuridine (BrdU; D) assays (n=6 in each group). * P <0.05 vs CM ‐Ad‐Control. † P <0.05 vs CM ‐Ad‐ FABP 4. E, Migration of HCASMC ‐ CM supplemented with 0.5% BSA in the absence and presence of 10 μg/mL FABP 4‐Ab for 15 hours assessed by scratch wound assay (n=6 in each group). * P <0.05 vs CM ‐Ad‐Control. † P <0.05 vs CM ‐Ad‐ FABP 4. F, Experimental design of HCASMCs coincubated with Ad‐ FABP 4– and Ad‐Control–transfected HCAECs using insert transparent wells for 24 hours ( HCASMC ‐ TW ). G and H, Gene expression of inflammatory cytokines (G) and proliferation‐ and adhesion‐related molecules (H) determined by quantitative real‐time PCR in HCASMC ‐ TW (n=6 in each group). * P <0.05 vs coincubation using transparent wells with Ad‐Control–overexpressed HCAECs ( TW ‐Ad‐Control). AU indicates arbitrary unit; IL, interleukin; Itga5, integrin a 5; Itgb3, integrin b 3; Mcp1, monocyte chemotactic protein‐1; Pdgfra, platelet‐derived growth factor receptor α; Pdgfrb, platelet‐derived growth factor receptor β; Tnfa, tumor necrosis factor α.

Article Snippet: HCAECs and HCASMCs were grown in Endothelial Cell Basal Medium‐2 (EBM‐2; Lonza) supplemented with EGM‐2 MV SingleQuots (Lonza) and in Smooth Muscle Cell Basal Medium (Lonza) supplemented with SmGM‐2 SingleQuots (Lonza), respectively, according to the manufacturer's instructions.

Techniques: Incubation, Plasmid Preparation, Binding Assay, Sequencing, Transfection, Expressing, Real-time Polymerase Chain Reaction, Migration, Scratch Wound Assay Assay, Derivative Assay

Journal: International Journal of Nanomedicine

Article Title: Human serum albumin-based probes for molecular targeting of macrophage scavenger receptors

doi: 10.2147/IJN.S197990

Figure Lengend Snippet: Evaluation of Mal-HSA-FITC uptake by different cell types. Fluorescence microscopy images of Mal-HSA-FITC probes (green) incubated for 3 h with ( A ) THP-1 macrophages, ( C ) HUVECs (EC), and ( D ) HCtSMCs (SMC). ( B ) Mal-HSA-FITC uptake by different cell types plotted against time. The results represent data from technical triplicates (9 images/well and timepoint) and are presented as mean and SEM. Data show significantly increased uptake of modified HSA probes by THP-1 cells compared with HUVEC (**** P <0.0001) and HCtSMCs ( #### P<0.0001) at 2.5 h and 3 h. None of the cell types recognized HSA-FITC (data not shown) (repeated measures two-way ANOVA (factors: cell type and time) and Tukey’s multiple comparison test). Abbreviations: HSA, human serum albumin; GCU, green calibration unit.

Article Snippet: Human carotid smooth muscle cells (HCtSMCs) (Cell Applications, San Diego, CA, USA) were cultured up to passage 7 in human smooth muscle cell media (Cell Applications).

Techniques: Fluorescence, Microscopy, Incubation, Modification, Comparison

Journal: Molecular cell

Article Title: Cicero predicts cis-regulatory DNA interactions from single cell chromatin accessibility data

doi: 10.1016/j.molcel.2018.06.044

Figure Lengend Snippet: Differentiating myoblasts follow similar single cell chromatin accessibility and gene expression trajectories. A) Single cell chromatin accessibility profiles for human skeletal muscle myoblasts (HSMM) were constructed with sci-ATAC-seq. Contaminating interstitial fibroblasts (common in HSMM cultures) were removed informatically prior to further analysis. B) Aggregated read coverage from sci-ATAC-seq experiments in the region surrounding TNNT1 and TNNI3 in myoblasts (0 hours) and myotubes (72 hours). Bulk ATAC-seq prepared from the same wells as experiment 2 are shown alongside DNase-seq from ENCODE for comparison (ENCODE experiments ENCSR000EOO and ENCSR000EOP (The ENCODE Project Consortium, 2012)). C) The single cell trajectory inferred from 2,725 myoblast sci-ATAC-seq profiles from experiment 1 by Monocle (see Methods). In subsequent panels and throughout the paper, we exclude cells on the branch to outcome F2 unless otherwise indicated. Inset shows the sc-RNA-seq trajectory reported for HSMMs (reproduced from Figure 2 of (Qiu et al., 2017a), cells were from the same lot and were cultured under identical conditions to those for sci-ATAC-seq). D) Distribution of cells in chromatin accessibility pseudotime from the root to trajectory outcome F1. E) Percent of differentiating cells whose promoters for selected genes are accessible across pseudotime. Black line indicates the pseudotime-dependent average from a smoothed binomial regression. F) Percent of cells whose promoters for selected genes in E are accessible in fibroblasts collected in growth medium (GM) or differentiation medium (DM), as well as myoblasts localized to the branch to F2. See also Figure S1.

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Skeletal Muscle Myoblasts (HSMM) HSMM derived from quadriceps biopsy (Lonza, catalog #CC-2580, lot #257130: healthy, age 17, female, of European ancestry, body mass index 19; cells were used within 5 passages of purchase) were cultured in skeletal muscle growth media (GM) using the SKGM-2 BulletKit (Lonza).

Techniques: Gene Expression, Construct, Comparison, RNA Sequencing, Cell Culture

Journal: Molecular cell

Article Title: Cicero predicts cis-regulatory DNA interactions from single cell chromatin accessibility data

doi: 10.1016/j.molcel.2018.06.044

Figure Lengend Snippet: Cicero constructs cis-regulatory models genome-wide from sci-ATAC-seq data. A) An overview of the Cicero algorithm (see Methods for details) B) Mean phastCons 46-way placental conservation scores of distal peaks connected to promoters. Peaks were stratified by distance from the promoter and coaccessibility score between the promoter and the distal peak. C) Mean distal site conservation score versus connected gene conservation score stratified by coaccessibility score. D) Odds ratios of concordant accessibility dynamics across differentiation in 54-1 myoblasts between pairs of sites that are coaccessible in HSMM. For each bin of coaccessibility in HSMM, pairs of peaks that overlapped peaks in 54-1 non-targeting controls were assessed for concordant dynamics (>2 log2 fold change in both peaks or < −2 log2 fold change in both peaks). Error bars indicate 95% confidence intervals calculated using Fisher’s exact test. Asterisks represent estimates significantly different than 1 (p-values < 0.05 by Fisher’s exact test). E) Two “phases” of myoblast differentiation illustrated. F) A summary of the Cicero coaccessibility links between the MYOG promoter and distal sites in the surrounding region. The height of connections indicates the magnitude of the Cicero coaccessibility score between the connected peaks. The top set of (red) links were constructed from cells in phase 1, while the bottom (in blue) were built from phase 2. See also Figures S3 and S4.

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Skeletal Muscle Myoblasts (HSMM) HSMM derived from quadriceps biopsy (Lonza, catalog #CC-2580, lot #257130: healthy, age 17, female, of European ancestry, body mass index 19; cells were used within 5 passages of purchase) were cultured in skeletal muscle growth media (GM) using the SKGM-2 BulletKit (Lonza).

Techniques: Construct, Genome Wide

Journal: Molecular cell

Article Title: Cicero predicts cis-regulatory DNA interactions from single cell chromatin accessibility data

doi: 10.1016/j.molcel.2018.06.044

Figure Lengend Snippet: MEIS1 and PBX1 knockout myoblasts fail to differentiate and show coordinated accessibility defects. A) Immunofluorescence microscopy images of non-template control, MEIS1 knockout and PBX1 knockout 54-1 cells at day 0 and day 7 post induction of differentiation. Nuclei are stained using DAPI, MYH3 is stained using anti-myosin MF20 and Alexa Fluor 594. B) Percent of peaks that open during differentiation in the NTC that fail to open in PBX1 and MEIS1 knockouts. Colors indicate the presence of MyoD binding in myoblasts and myotubes by ChIP-seq in HSMM. C) Odds ratios of concordant failure in accessibility gain across differentiation in 54-1 knockout myoblasts between pairs of sites that are coaccessible in HSMM. For each bin of coaccessibility in HSMM, pairs of peaks that overlapped peaks in 54-1 non-template controls were assessed for concordant failure to open (peaks that open in NTC but do not do so in knockouts). Color indicates knockout. Error bars indicate 95% confidence intervals calculated using Fisher’s exact test. Stars represent estimates significantly different than 1 (p-values < 0.05 by Fisher’s exact test). D) Similar to C. Odds ratios of concordant failure in accessibility gain given constitutive MyoD binding in one of the peaks. For each bin of coaccessibility in HSMM, pairs of peaks that overlapped peaks in 54-1 were assessed for presence of constitutive binding of MyoD in one or both of sites as well as coordinated failure to open. Only pairs of sites where both open in NTCs were included. Data is for MEIS1 knockout only due to a lack of sufficient power in PBX1. E) A model of how chromatin hub activation could be nucleated by a subset of “precociously” opening DNA elements. Such sites are occupied by TFs competent to bind relatively closed, inactive DNA elements, such as MEIS1, which may tether less competent factors such as MYOD to the hub. Subsequent recruitment of p300 and the BAF complex, possibly through intermediary factors (e.g. MYOD), leads to remodeling and acetylation of histones throughout the hub. These newly available sites are then bound by other transcriptional activators (e.g. MEF2), leading to the recruitment of Pol II. Moreover, acetylation of the histones downstream of assembled pre-initiation complexes reduces the barrier they pose to elongation, enhancing efficient transcription of genes within the hub.

Article Snippet: EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Skeletal Muscle Myoblasts (HSMM) HSMM derived from quadriceps biopsy (Lonza, catalog #CC-2580, lot #257130: healthy, age 17, female, of European ancestry, body mass index 19; cells were used within 5 passages of purchase) were cultured in skeletal muscle growth media (GM) using the SKGM-2 BulletKit (Lonza).

Techniques: Knock-Out, Immunofluorescence, Microscopy, Control, Staining, Binding Assay, ChIP-sequencing, Activation Assay