Journal: Molecular Therapy

Article Title: ZFN-Mediated In Vivo Genome Editing Corrects Murine Hurler Syndrome

doi: 10.1016/j.ymthe.2018.10.018

Figure Lengend Snippet: Decreased GAG Storage in ZFN+Donor-Treated MPS I Mice GAG levels present in the indicated tissues at 1 (A) or 4 (B) months post-treatment. Data are shown as mean ± SD. *p < 0.05, when compared to the gender-matched untreated MPS I group. n = 3 per group at 1 month and n = 4–5 per group at 4 months. (C) Levels of dermatan and heparan sulfate in mouse brain homogenates, 4 months post-treatment. Lines indicate mean of each group. All wild-type mice had levels of dermatan sulfate below the lower limit of quantification for this assay (<0.005 μg DS/mg protein lysate). *p < 0.05 for treated versus untreated male MPS I mice. For female MPS I mice, p = 0.0635 when comparing treated and untreated groups. (D) Images taken at 40× magnification from H&E-stained slides indicating the presence or absence of disease-related, highly vacuolated tissue-resident macrophages. Location of tissue resident macrophages indicated by black arrows.

Article Snippet: Mass spectrometry of brain tissue homogenates for levels of dermatan and heparan sulfate was performed at Pacific BioLabs (Hercules, CA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: Study workflow. H&E slides were annotated by a breast pathologist with regions defined as DCIS (blue) or IBC (red or orange). On a subsequent tissue section, slides were prepared for collagenase digestion to target the extracellular matrix (ECM). Mass spectrometry imaging (MSI) was performed with matrix-assisted laser desorption/ionization–quadrupole time-of-flight (MALDI-QTOF) imaging. Four samples were annotated per pathological lesion type and underwent the collagenase MSI workflow with high-resolution imaging at an individual lesion level. From the remaining eighteen-sample subset, specific slides were selected for further proteomic analysis multiplexing either tryptic or elastase digestion followed by mass spectrometry imaging. This schema was created in Biorender.com.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Mass Spectrometry, Imaging, Multiplexing

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: Spatial mapping of the extracellular proteome defines DCIS histopathology. ( A ) The eighteen-sample cohort underwent the workflow depicted, beginning with pathological annotation followed by extracellular matrix (ECM)-targeted mass spectrometry imaging and ECM peptide identification. ( B ) Spectra from pathologist-defined lesions with DCIS shown in blue and IDC shown in red demonstrate different relative peak intensity profiles. R. int. denotes the normalized relative intensity of peaks computed in mMass ® . ( C ) Hematoxylin and eosin-stained image of a mixed DCIS-IDC specimen demonstrates DCIS (blue) and IDC pathology (red). ( D ) Spatial segmentation analysis was used to define five main proteomic clusters. Cluster 1 (dark blue) annotates to adipocyte regions; Cluster 2 (green) defines borders between adipocyte and stroma; Cluster 3 (pink) localizes to stroma that includes DCIS lesions; Cluster 4 (blue) is localized to stroma and adipocytes primarily between tumor and adjacent tissue; Cluster 5 (yellow) annotates to the cancer region with diminishing detection distant from the tumor. ( E ) Pie chart depicting the proportion of peptide sequences identified from select protein classifications. Collagen fraction is further divided into collagen structural categories. ( F ) Spatial heat maps of a ColIα2 peptide depicted in red show distinct localization to DCIS lesions and surrounding ductal regions compared to the filamin-C peptide, which borders ductal regions and localizes to IDC. INPPL1 denotes inositol polyphosphate phosphatase like 1. Images were normalized to an internal peptide standard. Putative identifications were made by matching imaging data to an ECM database. Numbers following identification indicate the amino acid positions within the entire protein sequence. ( G ) Extracellular matrix peptides distinguished between DCIS, IDC, and DCIS-IDC. Heatmap is the average peptide expression detected across tissue images. ( H ) Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) of pathological regions depicts distinct clustering of regions by specimen classifications of DCIS ( n = 9), mixed DCIS-IDC ( n = 6), and IDC ( n = 4). ( I ) Loadings plot from sPLS-DA depicts the top ten peptide peaks that discriminate between specimen types. Ppm calculations between MALDI-QTOF imaging and LC-MS/MS were within 5 mass accuracy. sPLS-DA and heat map analyses were performed with MetaboAnalyst 5.0.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Histopathology, Mass Spectrometry, Imaging, Staining, Sequencing, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: DCIS specimens report distinct fibrillar collagen profiles to pathological regions. ( A ) A total of 43 extracellular matrix peptides were identified across tissue images distinguished between DCIS and IDC by an unpaired, two-tailed t -test ( p < 0.01). ( B ) A volcano plot of peaks identified via LC-MS/MS reports the most significantly differentially expressed peaks between DCIS and IDC pathologies. An absolute value fold change greater than 0.5 between DCIS and IDC with −log( p -value) greater than or equal to 1.5 is shown in orange if increased expression was found in IDC and blue if decreased expression was found in IDC. The volcano plot was created with VolcaNoseR. ( C ) Box-and-whiskers plots of fibrillar collagen sequences that are differentially expressed between DCIS ( n = 13) and IDC ( n = 10) lesions in eighteen samples by the Mann–Whitney test ( p < 0.05). ROC analyses of peaks adjacent to box-and-whiskers plots (AUROC > 0.75 and p < 0.05 by the Wilson/Brown t -test) are shown. Ox denotes oxidation, and HYP denotes hydroxylation of proline residues. ( D ) Location of the identified peptide within the protein sequence found to be differentially expressed by the Mann–Whitney test ( p < 0.05). ( E ) Spatial heatmaps of MALDI-QTOF imaging of 1084.498 m/z and 1458.700 m/z from two representative samples. Black annotations encircle IDC regions, while white annotations delineate DCIS regions. Ppm calculations between MALDI-QTOF imaging and LC-MS/MS were within 5 mass accuracy.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Expressing, MANN-WHITNEY, Sequencing, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: Serial enzymatic digest reveals pathology-specific proteomes and proteomic field cancerization. ( A ) Workflow for serial enzymatic digestion depicted with cellular localization from LC-MS/MS proteomic hits from each enzymatic digestion shown. Tissue was digested by collagenase to define stroma composition, trypsin to capture cellular features and additional extracellular composition, and elastase to target elastin. ( B ) Pathologist-defined lesions annotated by architectural pattern and distance to the invasive cancer site. ( C ) Segmentation analysis from 53 peptides derived from stroma. The tumor (yellow) and adjacent tissue (blue) represent distinct clusters with stromal composition extruding from the tumor (green). ( D ) DCIS lesions show distinct stromal signatures dependent on distance from IDC. ( E ) Spatial heatmaps of 3 collagenase peptide peaks (1291.664 m/z, 1458.701 m/z, 1588.781 m/z) depict discrete localization to IDC, DCIS, and surrounding normal adjacent tissue. Peptide sequences depicted within protein schemas. ( F ) Segmentation from serial tryptic digestion highlights the invasive cancer field (pink) and normal adjacent tissue (blue). Potential margins and punctate extensions form a unique cluster (green). ( G ) Differential expression detected by tryptic peptides based on location relative to IDC. ( H ) Spatial heatmaps of 3 tryptic peptide peaks (958.566 m/z, 1797.841 m/z, 1550.809 m/z) depict discrete localization to IDC, DCIS, and surrounding normal adjacent tissue. Peptide sequences depicted within protein schemas. ( I ) Segmentation analysis of peptides derived from elastase digestion. The tumor field (yellow and green) extends further into the normal adjacent tissue (blue and purple) compared to tryptic segmentation profiles. ( J ) DCIS lesions show differential signatures derived from 393 peptides produced by elastase digestion dependent on distance from IDC. ( K ) Spatial heatmaps of 3 elastase peptide peaks (906.472 m/z, 854.462 m/z, 1240.669 m/z) identified from an elastase-digested peptide library depict discrete localization to IDC, DCIS, and surrounding normal adjacent tissue. Peptide sequences depicted within protein schemas. Ppm calculations between MALDI-QTOF imaging and LC-MS/MS were within 5 mass accuracy.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Quantitative Proteomics, Produced, Imaging

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: MV histone doublets are re-localized to viral factories (VFs) (A) Histone dimer pairs (H2B-H2A and H4-H3) within Eukarya were aligned against the doublet Marseilleviridae histones using HHpred’s multiple sequence alignment tool (ClustalΩ). Known α helices from the histone fold (HF) domain in Eukarya are dark-colored tubes (H2B, red; H2A, yellow; H4, green; H3, blue; and additional helices, gray). Predicted α helices in MV histones were generated using HHrped’s Quick 2D prediction web server (shown in lighter coloration) within the Marseilleviridae histone doublets. Known R-T pairs and DNA binding residues are shown in Eukarya histones along with their conservation within Marseilleviridae histones; additional predicted DNA-binding residues are shown (positions demonstrated by lollipops). (B) Light microscopy fluorescence images (scale bar, 10 μm) of A. castellanii cells transfected with GFP- A. castellanii -H2A, MV-H2B-H2A-GFP, and MV-H3-H4-GFP, non-infected and infected with MV at 4 h PI. While GFP- A. castellanii -H2A concentrates only in the nucleus (N) of the non-infected cells, MV-H2B-H2A-GFP and MV-H3-H4-GFP are scattered in the entire cell (including the nucleus). Upon virus infection, GFP- A. castellanii -H2A remains in the nucleus (yellow arrows), while MV-H2B-H2A-GFP and MV-H3-H4-GFP re-localize to the VF (cyan arrows). DAPI staining remains in the nucleus all along the infection, but the intense fluorescence in the late VF hides the staining of the nucleus at 4 h PI. See also Figure S1 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Sequencing, Generated, Binding Assay, Light Microscopy, Fluorescence, Transfection, Infection, Virus, Staining

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae , the host Acanthamoeba castellanii , and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP- A. castellanii -H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to and .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Labeling, Virus, Infection, Sequencing, Generated, Residue, Binding Assay, Light Microscopy, Fluorescence, Transfection, Staining

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Mass spectrometry proteomics of the purified MV virions

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Mass Spectrometry, Purification, Membrane

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: MV-histones form nucleosome-like particles (A) SDS-PAGE of purified Melbournevirus (MV) histone doublets. (B) Sucrose gradient sedimentation of MV NLPs with 147 or 207 bp DNA. The compositions of MV NLPs were analyzed by native- and SDS-PAGE. (C) MV-NLP and eNuc were reconstituted on Widom ‘601’ DNA and Melbournevirus native DNA, respectively. The MV-NLP and eNuc were heat treated at 37 and 55°C. (D) Native PAGE of reconstituted MV mini-NLP (mini H2B-H2A instead of H2B-H2A) with 147 bp DNA. (E) GraFix of MV-NLPs with 207 bp DNA (top two panels) and 147 bp DNA (bottom two panels. Native- and SDS- PAGE of the crosslinked MV-NLP fractions representing successful crosslinking, compared to the native MV-NLP input. (F) Representative AFM images: Samples were diluted in TCS buffer and applied to APTES coated mica, rinsed with water and imaged on a NanoWizard Sa with a TAP300-GD cantilever. Samples include 147 bp DNA only, eNuc 147 , MV-H4-H3 with 147 bp DNA, MV-NLP 147 , MV-NLP 207 and MV-NLP 207 GraFix. Related to Figure 3 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: SDS Page, Purification, Sedimentation, Clear Native PAGE

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: S values (S (20,W) ), frictional ratios (f/f0), and calculated molecular weights (including confidence intervals) of histone-DNA complexes derived from SV-AUC

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Derivative Assay, Molecular Weight

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Cryo-EM reveals that MV-histone doublets form nucleosome-like structures with asymmetrically extending DNA (A) Overview of MV-NLP 147 and electron density. The equivalent regions of MV H3, H4, H2A, and H2B are shown in blue, green, yellow, and red, respectively. (B) Overlay of MV-NLP 147 (blue) with eNuc (gray). Only 80 bp of DNA with associated histones are shown for clarity. Superhelix locations (SHLs) are numbered from 0 to 6 starting from the nucleosome dyad (φ). (C) Comparison of the DNA path of eNuc 147 (gray ribbon diagram), MV-NLP 147 (blue electron density), and MV-NLP 207 (green electron density). (D) Charged surface representation of the histones for MV-NLP 147 and eNuc 147 . Coordinates for eNuc 147 were taken from 3LZ0. See also A and S3B and .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: Comparison of MV-NLP and eNuc histone structures (A) Superposition of two MV-H4-H3 doublets (green and blue) with the eukaryotic (H3-H4) 2 tetramer in gray (left), and a close-up of MV-H4-H3 doublet connector (right). Interactions of the MV-H4 N-terminal tail with the nucleosome are also shown. (B) A comparison of the four-helix bundle structure formed by H4 and H2B. (C) Superposition of two MV-H2B-H2A doublets (in red and yellow) with eukaryotic H2A-H2B dimers in gray (left), and a close-up of the MV-H2B-H2A doublet connector and docking domain. See for detailed information. Additional possible configurations for both connectors are shown in Figure S4 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Comparison

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet: MV-nucleosome histone dimer arrangement and histone connectors (A) Description of ϕ, the pseudo-dihedral angle measuring the relative orientations of the H2B-H2A subunits, between MV-NLP and eNuc. Larger magnitude values correspond to a dimer arrangement that is angled further away from the dyad axis. Fictitious particle locations are shown as black spheres, and histones (H3 in blue, H4 in green, H2A in yellow, and H2B in red) and DNA (silver and gold ribbons) are semi-transparent to allow for better visibility of each particle’s location. (B) Overlay of de novo loop models with feasible conformations connecting H2B to H2A and (C) H4 to H3 in the MV-histone doublets. Loop configurations were generated using Modeler (v9.20) and then placed in the nucleosome context. After manually removing physically non-relevant (knots) conformations and clashes identified with a cutoff distance of 0.8 Å using CPPTRAJ of the Amber MD package (v18), the three best loops are shown in different colors (cross correlation range against the simulated map is 0.614-0.623). Related to Figure 5 .

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Generated

Journal: Cell

Article Title: Virus-encoded histone doublets are essential and form nucleosome-like structures

doi: 10.1016/j.cell.2021.06.032

Figure Lengend Snippet:

Article Snippet: The A. castellanii histone H2A (2) gene (ACA1_364730A) was amplified from Acanthamoeba castellanii (Douglas) Neff (ATCC 30010TM) genomic DNA and cloned into the same plasmid engineered to yield a N-terminally GFP-tagged protein.

Techniques: Virus, Recombinant, Transfection, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: (A) 8MOP interacts with three peptide regions within the ErbB2 catalytic kinase domain. Qualitative peptide identifications within the ErbB2 catalytic kinase domain following LC-MS/MS analysis of a streptavidin pull-down of biotinylated-8MOP bait (see ). The transmembrane domain is indicated (red diamond) and the five C-terminus tyrosine autophosphorylation sites are indicated (p). (B) Non-reducing Western blot analysis of the interaction of 8MOP with ErbB2. BT474 cells were treated with 800dye-8MOP (Promega) or with vehicle (0.01% DMSO) alone served as control for 48 hr and then exposed to UV irradiation (2J) prior to Western blot analysis. The image on the left shows the Western blot for ErbB2 (red). The image on the right shows the same membrane directly scanned for the presence of 800dye-8MOP (green), which overlays the ErbB2 signal. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Irradiation, Membrane

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: The growth and viability of BT474 and SKBR3 cells (top bar graphs) after being subjected to the indicated treatment conditions. The combination of PUVA plus neratinib: P <0.0005 (BT474 and SKBR3 cells). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. (B) Western blot analysis showing steady-state ErbB2, ErbB3, and phospho-Akt (S473) protein levels in BT474 and SKBR3 cells treated according to the indicated treatment conditions. Vehicle alone (0.01% DMSO) served as a control. Steady-state actin protein levels served as a control for equal loading of protein. The results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

doi: 10.1371/journal.pone.0088983

Figure Lengend Snippet: Top bar graph shows the results of the growth assays performed in T47D and stably transfected T47D cell line. T47D cells expressing p85 ErbB2 were pretreated with 5 µM lapatinib or 5 µM 8MOP for 4 hr followed by irradiation in a UV Stratalinker 1800 (Statagene). Cells transfected with empty vector (T47D/Vector), and those treated with vehicle alone (0.01% DMSO) served as controls. The effects of the treatments on cell growth and viability are shown in the bar graph. P<0.0071 (8MOP + UVA irradiation). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. Steady-state phospho-p85 ErbB2 protein levels (dotted arrow) and phospho-p185 ErbB2 (solid arrow) are shown by Western blot. Actin steady-state protein levels served as a control to ensure for equal loading of protein. Results are representative of three independent experiments.

Article Snippet: ErbB2+ (BT474; SKBR3) and ErbB2 negative (MCF-7; T47D) human breast cancer cell lines, and the human foreskin fibroblast (HFF) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Stable Transfection, Transfection, Expressing, Irradiation, Plasmid Preparation, Western Blot, Control

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet:

Article Snippet: Jurkat cells, human immortalized T lymphocytes (ATCC, TIB-152, RRID: CVCL_0367) were maintained in RPMI 1640 medium (Sigma) supplemented with 10% (v/v) FBS and 1x Pen-Strep.

Techniques: Virus, Recombinant, Sequencing, Fluorescence, Modification, Protease Inhibitor, Polymer, Staining, Blocking Assay, Binding Assay, Activity Assay, Western Blot, Synthesized, Software, Control, Targeted Proteomics, Microscopy, Spectrophotometry, Mass Spectrometry