Journal: Cell Death & Disease

Article Title: NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance

doi: 10.1038/cddis.2016.175

Figure Lengend Snippet: NUPR1 regulates cell viability, growth, migration and invasion of HCC cells. ( a ) Cell viability of HCC cells transfected with siNUPR1 (siNUPR1 #1) and siNC was assessed by MTS assay after treatment with the indicated concentrations of sorafenib for 48 h. Data are expressed as the percentage of control cells and are the means±S.D. of three separate experiments, each performed in triplicate. * P <0.05, ** P <0.01. ( b ) Representative images of clonogenic assay of HCC cells transfected with siNUPR1 (siNUPR1 #1) and siNC. The experiment continued for 14 days. Surviving colonies were stained and counted. Data are expressed as the percentage of colonies and are the means±S.D. of three separate experiments, each performed in duplicate. ( c ) Representative images of wound-healing assay after NUPR1 siRNA-mediated gene silencing (shNUPR1 #1) in PLC/PRF/5. The experiment was conducted for 24 h. Data are reported as the percentage of cell migration and represent the average±S.D. of three experiments, each performed in duplicate. * P <0.05. ( d ) Representative images of transwell migration assay of Hep3B shNUPR1 (shNUPR1 #1) or Hep3B pSilencer cells. Data are reported as the percentage of migrated cells compared with control (siNC) and are the means±S.D. of three separate experiments, each performed in duplicate (* P <0.05). ( e ) Matrigel invasion assay in Hep3B shNUPR1 cells (shNUPR #1) compared with pSilencer as control. Data are reported as the percentage of invaded cells compared with control (pSilencer) and are the means±S.D. of three separate experiments, each performed in duplicate. * P <0.05

Article Snippet: A total of 10 × 10 6 Hep3B cells stably expressing shRNA against NUPR1 or control shRNA (pSilencer) were implanted subcutaneously in female nude athymic mice (Fox1 nu/nu) (6 weeks old, male; n =12) obtained from Envigo (Udine, Italy).

Techniques: Migration, Transfection, MTS Assay, Control, Clonogenic Assay, Staining, Wound Healing Assay, Transwell Migration Assay, Invasion Assay

Journal: Cell Death & Disease

Article Title: NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance

doi: 10.1038/cddis.2016.175

Figure Lengend Snippet: NUPR1 regulates expression of RELB and IER3 genes, and RELB and IER3 regulate cell viability and the colony-formation capacity of HCC cells. ( a ) Gene expression analysis by qPCR in HCC cells after NUPR1 gene silencing (siNUPR1 #1). Data are reported as the percentage of gene expression inhibition of each gene and are the means±S.D. of three separate experiments, each performed in triplicate. ( b ) Gene expression analysis, by qPCR, in Hep3B shNUPR1 (shNUPR1 #1) cells compared with pSilencer, as control. Data are expressed as reported in panel ( a ). ( c ) Western blotting analysis of P -ERK1/2 (Thr202/Tyr204) and total ERK1/2 in Hep3B shNUPR1 (shNUPR #1) and in control cells (pSilencer). ( d ) Gene expression analyses by qPCR after RELB and IER3 gene silencing in Hep3B cells. Data are expressed as reported in panel ( a ). ( e ) Cell viability of Hep3B cells transfected with siRELB, siIER3 and siNC was assessed by MTS assay after treatment with the indicated sorafenib concentrations for 48 h. Data are expressed as reported in . * P <0.05. ( f ) Representative images of the clonogenic assay of Hep3B cells transfected with siRELB, siIER3 and siNC. The experiment continued for 14 days. Surviving colonies were stained and counted. Data are expressed as reported in . * P <0.05; ns=non-significant

Article Snippet: A total of 10 × 10 6 Hep3B cells stably expressing shRNA against NUPR1 or control shRNA (pSilencer) were implanted subcutaneously in female nude athymic mice (Fox1 nu/nu) (6 weeks old, male; n =12) obtained from Envigo (Udine, Italy).

Techniques: Expressing, Gene Expression, Inhibition, Control, Western Blot, Transfection, MTS Assay, Clonogenic Assay, Staining

Journal: Cell Death & Disease

Article Title: NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance

doi: 10.1038/cddis.2016.175

Figure Lengend Snippet: NUPR1 knockdown inhibited tumor growth of Hep3B cells in nude mice. ( a ) Microphotography of 8 out of the 12 mice inoculated with stable Hep3B cells harboring NUPR1 shRNA (shNUPR1) (right flank) or non-specific shRNA (pSilencer) (left flank). ( b ) Microphotographs of tumors collected after 4 weeks of injection with Hpe3B pSilencer cells. ( c ) Tumor growth of Hep3B cells harboring pSilencer or shNUPR1. * P <0.05; ** P <0.01

Article Snippet: A total of 10 × 10 6 Hep3B cells stably expressing shRNA against NUPR1 or control shRNA (pSilencer) were implanted subcutaneously in female nude athymic mice (Fox1 nu/nu) (6 weeks old, male; n =12) obtained from Envigo (Udine, Italy).

Techniques: Knockdown, shRNA, Injection

Journal: Cell Death & Disease

Article Title: NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance

doi: 10.1038/cddis.2016.175

Figure Lengend Snippet: Functional analysis for the data set of differentially expressed genes and network analysis of dynamic gene expression obtained following shRNA-mediated NUPR 1 knockdown in Hep3B cells. ( a and b ) IPA functional pathway analyses of genes differentially expressed (≥ 3-fold) in Hep3B cells upon NUPR1 suppression. Top functions that meet a P -value cutoff of 0.05 are displayed. The orange line represents the cutoff value for significance. ( a ) Genes that were upregulated and ( b ) genes downregulated. ( c ) The five top-scoring networks were merged and are displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Intensity of the node color indicates the degree of up regulation (red) or downregulation (green). Nodes are displayed using various shapes that represent the functional class of the gene product (rhomboid=transporter; square=cytokine; diamond=enzyme; vertical oval=transmembrane receptor; horizontal oval=transcription factor; rectangle=nuclear receptor; hexagon=translation factor; circle=other). Edges are displayed with various labels that describe the nature of the relationship between the nodes:→acts on; -— binding only. The length of an edge reflects the evidence supporting the specific node-to-node relationship, as edges supported by articles from the literature are shorter. Dotted edges represent indirect interaction

Article Snippet: A total of 10 × 10 6 Hep3B cells stably expressing shRNA against NUPR1 or control shRNA (pSilencer) were implanted subcutaneously in female nude athymic mice (Fox1 nu/nu) (6 weeks old, male; n =12) obtained from Envigo (Udine, Italy).

Techniques: Functional Assay, Gene Expression, shRNA, Knockdown, Binding Assay

Journal: Cell Death & Disease

Article Title: NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance

doi: 10.1038/cddis.2016.175

Figure Lengend Snippet: RUNX2 regulates cell viability, growth, migration and the expression of NUPR1 , RELB and IER3 genes in Hep3B cells and is expressed in human HCC samples. ( a ) Gene expression analysis by qPCR after NUPR1 gene silencing (shNUPR1 #1) in Hep3B cells. ( b ) Colony assay after RUNX2 siRNA-mediated gene knockdown. Data are expressed as reported in . ( c ) Representative images of transwell migration assay after RUNX2 gene silencing in Hep3B cells. Data are expressed as reported in .** P <0.01. ( d ) Cell viability of Hep3B cells transfected with siRUNX2 (siRUNX2 #1) and siNC was assessed by MTS assay after treatment with the indicated concentrations of sorafenib for 48 h. Data are expressed as reported in . ( e ) Gene expression analysis after RUNX2 gene silencing (siRUNX2 #1) in Hep3B cells performed by qPCR. Data are expressed as reported in . ( f ) RUNX2 gene expression analysis in 12 HCC tissues performed by qPCR. Data are indicated as reported in . ( g ) RUNX2 protein expression levels were examined by immunohistochemistry in the NL ( A ) and HCC tissues ( B ). Magnification= × 20, insert magnification= × 40. Scale bar=100 μ m

Article Snippet: A total of 10 × 10 6 Hep3B cells stably expressing shRNA against NUPR1 or control shRNA (pSilencer) were implanted subcutaneously in female nude athymic mice (Fox1 nu/nu) (6 weeks old, male; n =12) obtained from Envigo (Udine, Italy).

Techniques: Migration, Expressing, Gene Expression, Colony Assay, Knockdown, Transwell Migration Assay, Transfection, MTS Assay, Immunohistochemistry

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