Journal: Aging Cell

Article Title: Liver osteopontin is required to prevent the progression of age‐related nonalcoholic fatty liver disease

doi: 10.1111/acel.13183

Figure Lengend Snippet: Liver OPN is p53 regulated. (a) Protein levels of p53 were evaluated by immunohistochemistry in liver sections of 3‐, 10‐, and 20‐month‐old (m) wild‐type (WT) mice ( n = 3–5). (b) OPN protein levels from liver homogenates were measured in WT and p53‐KO male and female mice fed a chow diet (CD) and a high‐fat diet (HFD) by immunoblotting using glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as loading control ( n = 4–6). (c) Liver p53 and OPN protein levels were evaluated in WT mice fed a HFD injected with p53‐dominant positive adenovirus (adp53) and GFP (adGFP), using transferrin or GAPDH as a loading control ( n = 4–6). (d) Protein levels of p53 and OPN in siCtrl and sip53 HepG2 cells were measured by immunoblotting using GAPDH as loading control. Extracellular OPN was measured using an ELISA ( n = 4–5). (e) OPN protein levels from HepG2 cells silenced for p53 and Hep3B cells treated with palbociclib (Palbo) were measured by immunoblotting using GAPDH as loading control. OPN media levels from Hep3B cells treated either with vehicle (Veh) or with palbociclib (Palbo) were measured by ELISA ( n = 4–8). Values are means ± SEM . Significant differences are denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001 (Student's t test)

Article Snippet: Data obtained from the Cell Line Encyclopedia (© 2019 The Broad Institute of MIT & Harvard) showed that the cell line with the least p53 expression, the Hep3B, also had the lowest OPN expression.

Techniques: Immunohistochemistry, Western Blot, Control, Injection, Enzyme-linked Immunosorbent Assay

Journal: Biomimetics

Article Title: Anticancer Activity of Thiophene Carboxamide Derivatives as CA-4 Biomimetics: Synthesis, Biological Potency, 3D Spheroid Model, and Molecular Dynamics Simulation

doi: 10.3390/biomimetics7040247

Figure Lengend Snippet: IC 50 (µM) of phenyl-thiophene-carboxamide compounds ( 2a – 2e ) on several cell lines.

Article Snippet: Our results showed that the PSA of the most synthesized structures was biomimetic to CA-4, and similar chemical and biological properties were observed against Hep3B cancer cell line.

Techniques:

Journal: Biomimetics

Article Title: Anticancer Activity of Thiophene Carboxamide Derivatives as CA-4 Biomimetics: Synthesis, Biological Potency, 3D Spheroid Model, and Molecular Dynamics Simulation

doi: 10.3390/biomimetics7040247

Figure Lengend Snippet: RHA-3 ( 2b ) and RHA-6 ( 2e ) perturb 3D hepatocellular spheroids’ formation. Images of cluster/s formed by Hep3B hepatocellular carcinoma in presence of 2b (17 µg/mL) or 2e (17 µg/mL) after 24 h of treatment compared to controls ( A ). Cluster percentage of occupied area relative to the negative control ( B ), cluster circularity ( C ), and cluster count ( D ). The non-treated cells are referred to as a negative control. At 100 μg/mL, DOX was utilized as a positive control. The scale bar represents a distance of 10 μm. Circularity scale: a value of 1 represents a perfect circle (ns: p > 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, and ****: p ≤ 0.0001).

Article Snippet: Our results showed that the PSA of the most synthesized structures was biomimetic to CA-4, and similar chemical and biological properties were observed against Hep3B cancer cell line.

Techniques: Negative Control, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: A biallelic mutation in IL6ST encoding the GP130 co-receptor causes immunodeficiency and craniosynostosis

doi: 10.1084/jem.20161810

Figure Lengend Snippet: The GP130 p.N404Y variant causes a defective acute phase response, and P1 primary fibroblasts can be rescued by lentiviral transduction of WT GP130. (A) Cellular and biochemical response to recurrent chest infections and cellulitis in patient GP130 p.N404Y. White blood cell count (WBC), neutrophils, CRP, and fibrinogen measurements are shown at different time points. Black/red lines indicate 3rd/97th percentile or lower/upper limit of the normal range, respectively. (B) CRISPR/Cas9-mediated KO of GP130 in human hepatoma Hep3B cells results in absent GP130 surface expression. (C) IL-6–mediated production of fibrinogen is GP130 dependent. Hep3B GP130-KO cells were stimulated with IL-6 for 24 h, and expression of the fibrinogen α chain ( FGA ; left) and fibrinogen β chain ( FGB ; right) was determined by quantitative PCR. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with unstimulated cells. Data represent summary results from four independent experiments (mean ± SEM). (D) Hep3B GP130-KO cells were transiently transfected with GP130 WT, GP130 p.N404Y-mutant plasmid, and FGA (left) and FGB (right) gene expression was analyzed after 24 h of IL-6 stimulation. Gene expression was determined relative to the housekeeping gene RPLP0 and expressed as fold-induction compared with the unstimulated vector control. Data represent pooled results from four independent experiments (mean ± SEM). (E) Fibroblasts of a healthy donor and P1 were stimulated with the indicated concentrations (ng/ml) of IL-6, IL-11, IL-27, OSM, and LIF. The levels of phospho-STAT3 (p-STAT3) were determined after 15-min stimulation by Phosflow. Titration curves are representative of two independent experiments. Curves are fitted by nonlinear/linear regression analysis. (F) Healthy donor and patient GP130 p.N404Y fibroblasts were treated as in E, and STAT1 phosphorylation (p-STAT1) was analyzed using flow cytometry. Titration curves are representative of two independent experiements. MFI, mean fluorescence intensity. (G) Lentiviral transduction of GP130 WT reconstitutes STAT3 phosphorylation in primary fibroblasts with p.N404Y variant. Fibroblasts were stimulated with 30 ng/ml IL-6 and 50 ng/ml IL-11 for 15 min. Quantification is based on four independent experiments. HD, healthy donor; LV, lentiviral. Differences were determined by Mann-Whitney U test. *, P < 0.05.

Article Snippet: Hep3B cells were obtained from the European Collection of Authenticated Cell Cultures and maintained in DMEM (Sigma-Aldrich) supplemented with 10% FCS.

Techniques: Variant Assay, Transduction, Cell Counting, CRISPR, Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Transfection, Mutagenesis, Plasmid Preparation, Control, Titration, Phospho-proteomics, Flow Cytometry, Fluorescence, MANN-WHITNEY