Journal: bioRxiv

Article Title: Semaphorin 3A and 3F overexpression in TIE2 hyperactive endothelial cells contribute to the pathological lumen expansion in venous malformation

doi: 10.1101/2025.07.18.665640

Figure Lengend Snippet: (a) Volcano plot of DEGs in TIE2-L914F compared to TIE2-WT EC. A total of 860 genes showed increased expression while 1118 genes showed decreased expression. Upregulated gene expression of Sema3A and Sema3F are highlighted. (b) Gene ontology analysis of RNA sequencing of TIE2-mutant EC compared to TIE2-WT EC. In TIE2-mutant EC the following molecular functions were amongst the top 25 upregulated: Chemorepellent activity, Semaphorin receptor binding and Neuropilin binding. (c) Immmunoblot of Sema3F and Sema3A in WT-EC, TIE2-WT and TIE2-L914F EC. (d) Quantitative ELISA assay of Sema3A and (e) Sema3F protein levels in WT-EC, TIE2-WT and TIE2-L914F cells. Mean±SD, one-way ANOVA, p-values are indicated. (f) RNA Scope assay showing Sema3F (cyan) and Sema3A (magenta) expression in patient dervied tissue sections. RNA scope was combined with UEA1 fluorescent staining to identify vessels. The expression in VM lesional vessels was compared to non-lesional vessels. Scale bar: 50µm. (g) Spatial gene expression levels were quantified and expressed as % of positive signal (coverage)/nucleus. n≥100 EC nuclei/group. Mean±SD, Welch’s t-test. P-values are indicated. (h) qPCR for Sema3F and Sema3A in control EC (HUVEC and ECFC) and four VM patient-derived EC (TIE2 p.L914F mutation). Mean, quartiles, Welch’s t-test. P-values are indicated.

Article Snippet: For co-culture assays, EC were additionally transduced with lentiviral constructs encoding for either red fluorescent protein (mCherry, Addgene, cat#36084) or green fluorescent protein (GFP) (Addgene, cat#17446-LV, ).

Techniques: Expressing, Gene Expression, RNA Sequencing, Mutagenesis, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, RNAscope, Staining, Control, Derivative Assay

Journal: bioRxiv

Article Title: A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

doi: 10.1101/2024.08.05.606664

Figure Lengend Snippet: 1A . Overview of RAW264.7-IRC cell line creation. The inflammatory reporter construct, created using Gibson cloning, contained an mCherry fluorescent reporter under an inflammatory promoter, termed synthesis friendly inflammatory promoter (SFNp) , and was encapsulated and transduced via lentivirus. The cells then underwent puromycin selection to create a homogenous population of cells. RAW264.7-IRCs were then stimulated using different cytokines and inflammatory agents to induce mCherry expression. 1B . LPS dose curve showing range of mCherry fluorescence geometric mean values measured using flow cytometry across a range of LPS doses from 0.001 to 100 ng/mL. Individual data points are plotted for each biological replicate. Each dose was done in triplicate. Trend line calculated using the three-parameter dose-response curve, which yielded an EC50 = 0.61 ng/mL with an R 2 value of 0.94. Dose-response curve line equation can be found in Table S1 . 1C . TNF-α concentration levels (pg/mL) in cell culture supernatant measured using ELISA. Cells were treated with LPS at concentrations ranging from 0.001 to 1 ng/mL for 24 hours. Each data point represents an average of 2 technical replicates. Trend line calculated using the three-parameter dose-response curve, which yielded an EC50 = 0.87 ng/mL with an R 2 value of 0.987. Dose-response curve line equation can be found in Table S2. 1D . Plot of mCherry fluorescent geometric mean values plotted against respective TNF-α concentration levels (pg/mL). X -values represent the average of the 3 biological replicates of TNF-α concentration (from ) and y -values represent the average of the 3 biological replicates of mCherry geometric mean (from ) ± SD. Trend line calculated using linear regression, y = 21.12 x -8156, with an R 2 value of 0.90. Linear regression parameters can be found in Table S3. 1E . RAW264.7-IRC responsiveness. RAW264.7-IRCs were treated with LPS and IFN-γ for 24 hours, 24h cells were harvested for flow cytometry. Stimulation media was replaced with fresh media for the 48h and 72h samples. 24h post-removal of stimulation media, the 48h samples were harvested for flow cytometry, and the same was done for the 72h samples. Mean ± SD, with individual data points for each biological replicate. (**** p-value ≤ 0.0001, *** p-value ≤ 0.001, ** p-value ≤ 0.01, n.s. p-value > 0.05). 1F . Timeline of treatment for RAW264.7-IRC responsiveness.

Article Snippet: The mCherry red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Construct, Cloning, Selection, Expressing, Fluorescence, Flow Cytometry, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

doi: 10.1101/2024.08.05.606664

Figure Lengend Snippet: 2A . Phenotyping via immunostaining of RAW264.7-IRCs for CD206 (green), a common anti-inflammatory marker, and CD86 (blue), a common inflammatory marker, co-localized with mCherry expression between no treatment and LPS treated cells (10ng/mL). Scale bars are 20 μm. 2B . Co-localization of CD86 and CD206 with mCherry expression in non-stimulated RAW264.7-IRCs. 2C . Co-localization of CD86 and CD206 with mCherry expression in LPS-stimulated RAW264.7-IRCs. 2D. Ratio of mean fluorescent intensities of CD86:CD206 to mCherry fluorescent intensity.

Article Snippet: The mCherry red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Immunostaining, Marker, Expressing

Journal: bioRxiv

Article Title: A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

doi: 10.1101/2024.08.05.606664

Figure Lengend Snippet: 2A . Evaluation of viral and non-viral delivery methods using a dual-reporter assay at 24 and 48 hours post-delivery. Efficiency of delivery and inflammatory response were determined by using different viral (AAV 1, 2, 5, 8, 9 and Ad5/35) and non-viral (TransIT-X2 and Lipofectamine 2000) delivery methods carrying a GFP payload to RAW264.7-IRCs at timepoints of 24 hours and 48 hours post-delivery. %GFP+ representing efficient delivery and %mCherry+ representing inflammatory response. Percentages represent the average of 3 biological replicates. Each bar represents the average percentages from 3 biological replicates. TransIT-X2 and Lipofectamine 2000 were only evaluated at 48 hours post-transfection per manufacturer protocol. Raw data points can be found in Table S4. 2B . Evaluation of the geometric mean of GFP to mCherry at 24 and 48 hours post-delivery. The data represented here is the same samples in 2A. Individual data points were plotted for each biological replicate. Y-axes between 24 and 48 hours is different. Raw data points can be found in Table S4. Statistics done using One-Way Anova and Tukey’s Post-Hoc Analysis can be found in Table S5 for 24-hour and S6 for 48-hour. 2C . Representative cell populations from dual-fluorescence reporter assay. Populations represented here are RAW264.7 cells with no reporter to set the negative control for GFP/mCherry, non-treated RAW264.7-IRCS (NT) to determine basal mCherry expression among reporter cells, and then three representative populations from RAW264.7-IRCs treated with AAV 1, Ad 5/35, and TransIT-X2, respectively. Gating strategy for dual-fluorescence assay can be found in Figure S2.

Article Snippet: The mCherry red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Reporter Assay, Transfection, Fluorescence, Negative Control, Expressing

Journal: PLOS Biology

Article Title: Yersinia pestis can infect the Pawlowsky glands of human body lice and be transmitted by louse bite

doi: 10.1371/journal.pbio.3002625

Figure Lengend Snippet: Fluorescence microscopy images of KIM6+(pmCherry) Y . pestis localization patterns observed in body lice following infection: (A) mCherry (orange or red) negative (no infection detected) (B) midgut only infection ( C) 1 or 2 foci of infection in the head (yellow arrows; with or without bacteria simultaneously detected in the midgut) or (D) bacteremic (bacteria detected in the hemocoel). (E) Cropped and enlarged image of the head of infected louse in panel C. MG = Midgut, H = Head, SD = Stomach disc. The louse exoskeleton and SD are autofluorescent. Scale bars = 200 μm. (F) The frequency of infection patterns observed ( n = 35–40 lice) from the direct (top) or delayed (bottom) groups for 1 week of infection. Vertical bars show the standard error of the mean for 3 independent experiments. (G) Diagram of a transverse section of the head of a body louse based on our histology and . Major anatomical structures include: the pharynx (Ph), the Pawlowsky glands (PGs), the PG ducts (PDs), and the chamber housing the mouthparts (MC). (H) Immunohistochemical detection of Y . pestis infection (red arrows; purple/pink staining) in the PGs and ducts that lead to the MC from a louse diagnosed with foci of infection in the head. Scale bar = 20 μm. (I) Absence of Y . pestis staining in an uninfected control louse. Images are representative of 5 μm sections of interest acquired from 4 infected and control lice. IHC images are counterstained with hematoxylin (blue). Summary data for this figure can be found in .

Article Snippet: Yersinia pestis , Yersinia pseudotuberculosis , or Escherichia coli strains ( ) transformed with a plasmid constitutively expressing the mCherry red fluorescent protein (pMcherry; Takara Bio) were streaked for isolation on blood agar plates (5% sheep’s blood), incubated for 24 ( E . coli and Y . pseudotuberculosis ) or 48 h ( Y . pestis ) at 28°C, and a single large colony was selected to inoculate 100 ml of brain heart infusion (BHI) broth supplemented with 10 μg/ml hemin and 100 μg/ml carbenicillin.

Techniques: Fluorescence, Microscopy, Infection, Bacteria, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

doi: 10.1371/journal.pone.0043772

Figure Lengend Snippet: Bacterial strains and plasmid constructs used in this study.

Article Snippet: pmCherry , Clonetech ( http://www.clontech.com/ ) , pUC, mCherry red fluorescence protein, Amp R.

Techniques: Plasmid Preparation, Construct, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: One for All or All for One: Heterogeneous Expression and Host Cell Lysis Are Key to Gene Transfer Agent Activity in Rhodobacter capsulatus

doi: 10.1371/journal.pone.0043772

Figure Lengend Snippet: Representation of the puf operon and RcGTA promoter regions fused to the mCherry gene, showing the fluorescent protein modified N-terminal coding sequence translated from the pufB or RcGTA g1 start codon. The bent arrow indicates either the puf operon or RcGTA promoter. The start codons of the pufB , RcGTA g1 , and mCherry genes are shown in bold, separated by eight codons from the plasmid pmCherry multiple cloning site. Positions of restriction sites introduced are indicated as Hind III ( H ), BamH I ( B, and sequence underlined ) and EcoR I ( E ). B. A broad wavelength absorbance scan of R. capsulatus SB1003 is shown as a black trace. The emission spectra of eGFP and mCherry are represented as green and red lines, respectively.

Article Snippet: pmCherry , Clonetech ( http://www.clontech.com/ ) , pUC, mCherry red fluorescence protein, Amp R.

Techniques: Modification, Sequencing, Plasmid Preparation, Clone Assay