Journal: Journal of Biomedical Science

Article Title: Extracellular arginine availability modulates eIF2α O-GlcNAcylation and heme oxygenase 1 translation for cellular homeostasis

doi: 10.1186/s12929-023-00924-4

Figure Lengend Snippet: The O-GlcNAcylation of eIF2α downregulates the antioxidant capacity, cell recovery and migratory ability of breast cancer cells in response to ArgS. A Immunoprecipitation coupled with immunoblot analysis was performed to measure the levels of GlcNAz-modified (O-GlcNAcylated) proteins in parental HEK 293T cells (n = 3; left panel ). The O-GlcNAz-modified eIF2α level was calculated by normalizing the densitometric tracing of eIF2α signal to the H3 signal ( right panel ). The values were quantified using ImageLAB software and adjusted for background. B The same analysis was performed in WT and O-GlcN-mut, eIF2α overexpressing HEK 293 T cells (n = 2; left panel ). The O-GlcNAz-modified eIF2α level was calculated as described in A , and the ratio in each experimental condition was compared to the reference (WT + Arg, set to 1; left panel ). One representative immunoblot is shown for each analysis, with indicated antibodies. The black arrowheads indicate endogenous eIF2α, and the white arrowheads indicate FLAG-tagged eIF2α. C The levels of ROS were measured in BT-549 cells stably expressing WT or O-GlcN-mut eIF2α after 48 h of ArgS treatment (n = 3). The ROS levels were quantified using CellROX™ and flow cytometry. D The relative cell recovery was monitored in BT-549 cells stably overexpressing WT or O-GlcN-mut eIF2α over a 5-day period, after 24 h of ArgS treatment and replacement with complete medium. The cell recovery was determined using ACP assays and the data were normalized to the values of Day 0, set to 1. E The cell migration was measured in BT-549 cells stably overexpressing WT or O-GlcN-mut eIF2α after 24 h of ArgS treatment and replacement with complete medium. The cell migration was determined by calculating the % confluency within the gap area and was imaged and quantified using Image J software at 0, 12, 24, and 30 h. Data are presented as mean ± s.e.m. and analyzed using Two-Way ANOVA followed by Tukey's multiple comparison test. Statistical significance was determined with * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Human triple-negative breast cancer BT-549, MDA-MB-231, Hs578T, MDA-MB-468, and MDA-MB-435, human estrogen receptor- and progesterone receptor-positive breast cancer MCF7, and human embryonic kidney (HEK) 293T (HEK293 cells with SV40 T-antigen) cells were originally purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Recovery, Immunoprecipitation, Western Blot, Modification, Software, Stable Transfection, Expressing, Flow Cytometry, Migration, Comparison

Journal: Acta biomaterialia

Article Title: Two-photon Polymerized Poly(caprolactone) Retinal Cell Delivery Scaffolds and their Systemic and Retinal Biocompatibility

doi: 10.1016/j.actbio.2019.04.057

Figure Lengend Snippet: Scaled-up prototype of crosslinked PCLTA 900 in the degenerating pig sub-retinal space 30 days after transplantation. Representative en face (A) and horizontal B-scan (B) optical coherence tomographs (green line in A indicates cross-sectional plane shown in B) of the post-mortem Pro23His pig eye show placement of the transplanted polymer (border indicated with arrows in A and marked with * in B). Representative histological (C and D) and immunohistochemical (E and F) images of retinal sections (RPE: retinal pigment epithelium; Ph: photoreceptor cells; INL: inner-nuclear layer; GCL: ganglion cell layer) show the embedded PCL scaffolds (marked with * and in E and F also autofluorescent in green) and surrounding tissue with some presumptive RPE cells (pigmented, shown with arrow in D) and photoreceptor cells (Recoverin positive, shown with arrows in F) infiltrating the scaffold. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For immunohistochemistry, tissues were treated with anti-recoverin antibody (Millipore, 1:250) to detect photoreceptor cells and DAPI for cell nuclei.

Techniques: Transplantation Assay, Immunohistochemical staining

Journal: EClinicalMedicine

Article Title: Upregulation of pulmonary tissue factor, loss of thrombomodulin and immunothrombosis in SARS-CoV-2 infection

doi: 10.1016/j.eclinm.2021.101069

Figure Lengend Snippet: Anti-thrombin/Factor VIIa (AT/VIIa) complex, and microparticle-TF (MP-TF) levels in SARS-CoV-2 infection and controls. (A) AT/VIIa complex and (B) MP-TF plasma levels were determined by ELISA in healthy controls and patients with COVID-19 patients. Ixolaris, a TF inhibitor, blocks MP-associated TF activity. Each symbol correspond one patient. Shaded bars indicate the means, and the lines show the SEM. (C) Ixolaris prolongs the PT, but not the aPTT. (D) Thrombin generation assay. Reactions were initiated by the addition of CaCl 2 , phospholipids and TF, with and without Ixolaris, as indicated. Each curve shows the mean of triplicate reactions in pooled plasma. NS, non-significant; ** p < 0.01, **** p < 0.0001 (Kruskall-Wallis test).

Article Snippet: Zymogen Microparticle-Tissue Factor (MP-TF) was purchased from Hyphen Biomed/Aniara Diagnostica (West Chester, OH).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Activity Assay