Journal: Molecular cell

Article Title: Heat Shock Factor 1 Is a Direct Antagonist of AMP-Activated Protein Kinase

doi: 10.1016/j.molcel.2019.08.021

Figure Lengend Snippet: Key Resources Table

Article Snippet: Peroxidase-AffiniPure goat anti-rat IgG (H+L) antibody , Jackson ImmunoResearch Labs , Cat# 112-035-003, RRID: AB_2338128.

Techniques: FLAG-tag, Amplification, Polymer, Staining, Plasmid Preparation, Virus, Recombinant, Transfection, Viability Assay, In Situ, Amplex Red Cholesterol Assay, Protease Inhibitor, Extraction, Bioassay, Sandwich ELISA, Mutagenesis, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Silver Staining, Mass Spectrometry, cDNA Synthesis, Western Blot, ChIP-qPCR, Sequencing, Luciferase, Software, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes

doi: 10.1093/nar/gkw105

Figure Lengend Snippet: The KMT2D complex interacts with the SPOC-domain of SHARP. ( A ) Schematic representation of the RBP-J/SHARP repressor complex. SHARP interacts with RBP-J via its RBP interaction domain and with NCoR/HDAC complexes due to its C-terminal SPOC-domain. ( B ) SPOCome. A biotinylation-tagging approach followed by mass spectrometry was performed in pre-T cells and allowed to identify the KMT2D and NCoR complexes as interactors of the SPOC domain of SHARP. A complete list of interactors is provided in Supplementary Table S1. ( C ) Bio-SPOC interacts with KMT2D complex specific component UTX but not with KMT2A in pre-T cells. Streptavidin magnetic beads were used to pull-down the SPOC interactors. Precipitated proteins were analyzed by Western blot using streptavidin-HRP and specific antibodies directed against RbBP5, UTX and KMT2A. ( D and E ) The SPOC domain of SHARP interacts with the C-terminus of KMT2D in vitro . ( D ) Only the KMT2D-7 construct interacts with GST-SPOC (lane 2). CtIP, a known binding partner of SPOC, served as a positive control. ( E ) Input of the cell free synthesized 35 S-labeled KMT2D fragments [(KMT2D-4, lane 1), (KMT2D-5, lane 2), (KMT2D-7, lane 3) and CtIP (lane 4, positive control) used in the GST pull-down experiments shown in Figure . ( F and G ) Mapping of the SPOC-KMT2D interaction in cellular extracts. HEK293 cells were transfected with the indicated expression constructs for Flag-tagged SPOC domain of SHARP and GFP-tagged KMT2D fragments. ( F ) GFP-KMT2D fragments 7b (lane 1) and 7d (lane 3) coimmunoprecipitate with the SPOC domain of SHARP. A weak interaction was detected with the KMT2D-7c fragment (lane 2), which represents the SET domain of KMT2D (see also Figure ). HEK293 cells were transfected with the indicated expression constructs for Flag-tagged SPOC domain of SHARP and GFP-tagged KMT2D fragments. The asterisk denotes the heavy chain of the antibody used for immunoprecipitation. ( G ) Input control of the proteins used in (F). ( H ) Schematic representation of the KMT2D constructs used in (D)–(G) and their binding capacity to the SPOC-domain. Amino acid numbering is according to accession NP_001028448.3. KMT2D domains: PHD-zf, PHD-zinc finger (CCD: 197604); PHD-f, PHD-finger (CCD: 201356); HMG, HMG-box (CCD: 28965); FYRN, F/Y-rich N-terminus (CCD: 191411); FYRC, F/Y rich C-terminus (CCD: 197781); SET, SET domain (CCD: 197640). ( I ) Endogenous RBP-J (left panel, lane 3) and KMT2D (right panel, lane 4) interact with SHARP in HeLa cells. The asterisk denotes the heavy chain of the antibody used for immunoprecipitation. ( J ) KMT2D (left) and SHARP (middle) are located in the nuclei of HeLa cells and show regions of colocalization (right) as determined by confocal microscopy. Scale bar, 20 μm.

Article Snippet: 5 mg of nuclear extract were incubated with 150 μl streptavidin magnetic beads (Dynabeads M-280, Invitrogen 112.06) for 2 h at 4°C.

Techniques: Mass Spectrometry, Magnetic Beads, Western Blot, In Vitro, Construct, Binding Assay, Positive Control, Synthesized, Labeling, Transfection, Expressing, Immunoprecipitation, Control, Confocal Microscopy

Journal: Journal of ethnopharmacology

Article Title: Inhibitory effects and mechanisms of the anti-covid-19 traditional Chinese prescription, Keguan-1, on acute lung injury.

doi: 10.1016/j.jep.2021.114838

Figure Lengend Snippet: Fig. 5. Effects of Keguan-1 on endothelial injury in the LPS-induced ALI mouse model. (A) The BALF protein concentration. (B) The BALF total cell count. (C) The levels of Evans blue extracted from lungs. (D) The concentration of Ang II in BALF. (E–H) The expression of ICAM-1 (E), claudin-5 (F), JAM-1 (G), and VE-cadherin (H) in mice lungs as determined by using western blotting. The data are presented as the mean ± SEM. #P < 0.05 and ##P < 0.01 vs. control; *P < 0.05 and **P < 0.01 vs. LPS group.

Article Snippet: LPS (Escherichia coli 055:B5) was purchased from Abbreviations TCM traditional Chinese medicine ARDS acute respiratory distress syndrome RCT randomised clinical trial LPS lipopolysaccharide ALI acute lung injury BALF bronchoalveolar lavage fluid COVID-19 coronavirus disease 19 SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 HPLC-MS high-performance liquid chromatography-tandem mass spectrometry DXM dexamethasone TNF-α tumour necrosis factor alpha IL-6 interleukin-6 IL-1α interleukin-1alpha IL-1β interleukin-1beta KC keratinocyte-derived chemokine MIP2 macrophage inflammatory protein 2 Ang II angiotensin II PBS phosphate-buffered saline PMSF phenylmethanesulfonyl fluoride PaCO2 CO2 partial pressure PaO2 O2 partial pressure SO2 oxygen saturation ACE2 angiotensin-converting enzyme II ICAM-1 intercellular adhesion molecule-1 Z. Bai et al. Journal of Ethnopharmacology 285 (2022) 114838 Sigma-Aldrich (St. Louis, MO, USA); dexamethasone (DXM), from HARVEYBIO (Beijing, China); Formalin fixative solution, from Yili Fine Chemicals Co., Ltd. (Beijing, China); the tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1α ELISA kits, from Dakewe Biotech Co., Ltd (Shenzhen, China); the IL-1β ELISA kit, from R&D Systems (Minneapolis, MN, USA); the keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) ELISA kits and Annexin V-PE/7-AAD apoptosis kit, from MultiSciences (Lianke) Biotech Co., Ltd (Hangzhou, China); the Angiotensin (Ang) II ELISA kit, from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); the BCA protein assay kit, from Thermo Fisher Scientific (Waltham, MA, USA); FITC anti-mouse Ly6G and APC anti-mouse F4/80 antibodies, from Biolegend (San Diego, CA, USA); intercellular adhesion molecule-1 (ICAM-1) and claudin-5 antibodies, from Abcam (Cambridge, UK); JAM-1, VE-cadherin, and GAPDH antibodies, from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and Evans blue stain, from Sigma-Aldrich.

Techniques: Protein Concentration, Cell Counting, Concentration Assay, Expressing, Western Blot, Control

Journal: Proteomics

Article Title: Rapid generation of functional nanovesicles from human trophectodermal cells for embryo attachment and outgrowth.

doi: 10.1002/pmic.202300056

Figure Lengend Snippet: FIGURE 3 Uptake of NVs and EVs by HEC1A endometrial cells. (A) Confocal fluorescent microscopy images demonstrating uptake of DiI lipophilic fluorescent dye labelled NVs or EVs (red) by HEC1A endometrial cells after 2 h incubation (n = 3). (B) Fluorescent Z-stack image displaying intracellular distribution of DiI-labelled NVs (red). Nuclei were stained with Hoechst. Scale bar 10 µm.

Article Snippet: Human endometrial carcinoma HEC1A cells (HTB-112) were pur- chased from American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Microscopy, Incubation, Staining

Journal: Proteomics

Article Title: Rapid generation of functional nanovesicles from human trophectodermal cells for embryo attachment and outgrowth.

doi: 10.1002/pmic.202300056

Figure Lengend Snippet: FIGURE 4 TSC-derived NVs and EVs enhance trophectodermal spheroid attachment to low receptive endometrial cells and mouse embryo outgrowth. (A) Experimental workflow for co-culture attachment assay. Created with Biorender. (B) Box plot indicating percentage of TSC-spheroid attachment to HEC1A endometrial cells following PBS control, NV, or EV treatment (n = 5). (C) Experimental workflow for mouse embryo fibronectin attachment and outgrowth assay. (D) Box plot indicating quantified area of mouse embryo outgrowth 72 h following PBS control, NV, or EV treatment. (E) Bright-field microscopic images of mouse embryos 72 h following PBS control, NV, or EV treatment (n = 4). Nuclei were stained with Hoechst. Scale bar 500 µm.

Article Snippet: Human endometrial carcinoma HEC1A cells (HTB-112) were pur- chased from American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Derivative Assay, Co-Culture Assay, Control, Staining

Journal: Proteomics

Article Title: Rapid generation of functional nanovesicles from human trophectodermal cells for embryo attachment and outgrowth.

doi: 10.1002/pmic.202300056

Figure Lengend Snippet: FIGURE 5 Proteome profiling of HEC1A cells treated with NVs, EVs, or PBS. (A) Workflow for mass spectrometry-based proteomic profiling, comprising sera-bead sample preparation with nano-liquid chromatography tandem mass spectrometry and data processing/informatics. Created with Biorender. (B) Principal component analysis of HEC1A cellular proteome treated with NVs, EVs, or PBS control (volume matched). (C) Venn diagram of proteins identified in HEC1A cells after treatment with NV, EV, or PBS. (D) Two-way scatter plot highlighted the most significantly upregulated and downregulated proteins in NV-treated and EV-treated HEC1A cells compared to PBS control. (E) EnrichmentMap of Gene Ontology (biological process) and Reactome processes overrepresented in significantly upregulated proteins in both NV-treated and EV-treated HEC1A cells compared to PBS control.

Article Snippet: Human endometrial carcinoma HEC1A cells (HTB-112) were pur- chased from American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Mass Spectrometry, Sample Prep, Liquid Chromatography, Control