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

Journal: International Journal of Biological Sciences

Article Title: Lnc-ZEB2-19 Inhibits the Progression and Lenvatinib Resistance of Hepatocellular Carcinoma by Attenuating the NF-κB Signaling Pathway through the TRA2A/RSPH14 Axis

doi: 10.7150/ijbs.85270

Figure Lengend Snippet: lnc-ZEB2-19 inhibits the progression and Lenvatinib resistance of HCC cells. In this figure, Hep3B and MHCC-97H cells overexpressing or knocking down lnc-ZEB2-19 and the control group were named Lv-ZEB2-19, Lv-Con, sh-Lnc, and sh-Con. (A) CCK8 assay was conducted to confirm the viability of each group of HCC cells. The effects of lnc-ZEB2-19 on HCC cell colony formation ability (B, C) and migration ability from wound healing assays (D) and invasion ability from Transwell assays (E, F) and sphere-formation ability (I) . Western blot assays were applied to verify the relative protein level of EMT (G, H) and stemness (K) . The sensibility of lenvatinib was validated through IC50 assays (J) .

Article Snippet: Hep3B cells in the lnc-ZEB2-19 overexpression and control groups were used for RNA sequencing screening (BGI Genomics Co., Ltd., Shenzhen, China).

Techniques: Control, CCK-8 Assay, Migration, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Novel compound cedrelone inhibits hepatocellular carcinoma progression via PBLD and Ras/Rap1

doi: 10.3892/etm.2019.8080

Figure Lengend Snippet: Cedrelone mediates cell bioactivities in hepatocellular carcinoma. (A) Chemical structure of cedrelone. (B) Cells were treated with different concentrations of cedrelone for 24 h and the growth of Hep3B and HepG2 cells was detected via sulforhodamine B staining. (C) Cells were treated with different concentrations of cedrelone for 24 h and a Cell Counting kit-8 assay was used to measure cell viability. (D) Cells were treated with different concentrations of cedrelone for 24 h and trypan blue staining was used to detect the cell death ratio. Cells were treated with 20 µM cedrelone for 24 h and cell, (E) detection of cell invasion and (F) statistical analysis, (G) detection the cell migration and (H) statistical analysis. Scale bar, 200 µm. (I) Epithelial-mesenchymal transition markers were detected via western blotting after cells were treated with 20 µM cedrelone for 24 h. (J) The cell apoptosis ratio was detected using a FACScan flow cytometer, the abscissa represents the level of Annexin V staining whilst the ordinate represents propidium iodide staining. (K) Quantified results from (J). (L) Apoptosis marker levels were detected via western blotting after cells were treated with 20 µM cedrelone for 24 h. In figs B, C and D, *P<0.05, **P<0.01 and ***P<0.005 vs. DMSO group. In figs A, E, F, G and H, ***P<0.005. c-, cleaved; DMSO, dimethyl sulfoxide; PARP, poly-ADP ribose polymerase.

Article Snippet: HepG2 and Hep3B cells (1×10 4 ) were incubated with different concentrations (5, 10, 20 and 40 μM) of cedrelone (Shanghai Topscience Co., Ltd.) for 24 h at 37°C with 5% CO 2 and 10% trichloroacetic acid was added.

Techniques: Staining, Cell Counting, Migration, Western Blot, Flow Cytometry, Marker

Journal: Experimental and Therapeutic Medicine

Article Title: Novel compound cedrelone inhibits hepatocellular carcinoma progression via PBLD and Ras/Rap1

doi: 10.3892/etm.2019.8080

Figure Lengend Snippet: PBLD expression is downregulated in HCC. (A) Immunohistochemistry was performed to detect PBLD expression in HCC and adjacent non-tumors tissue. Scale bar, 200 µm for all five images. (B) PBLD mRNA expression in tumor and non-tumor tissues was detected via qPCR. (C) PBLD mRNA expression in tumors tissue with different degrees of differentiation was detected by performing qCR. PBLD protein expression in (D) tumor tissues with different degrees of differentiation and (E) tumor and non-tumor tissues, as well as (F) different cell lines was detected via western blotting. HL-7702 is a normal hepatocyte cell line, whereas HepG2, Hep3B and Huh-7 are tumor cell lines. *P<0.05 and **P<0.01 as indicated. PBLD, phenazine biosynthesis-like domain-containing protein; HCC, hepatocellular carcinoma; qPCR, quantitative PCR.

Article Snippet: HepG2 and Hep3B cells (1×10 4 ) were incubated with different concentrations (5, 10, 20 and 40 μM) of cedrelone (Shanghai Topscience Co., Ltd.) for 24 h at 37°C with 5% CO 2 and 10% trichloroacetic acid was added.

Techniques: Expressing, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Novel compound cedrelone inhibits hepatocellular carcinoma progression via PBLD and Ras/Rap1

doi: 10.3892/etm.2019.8080

Figure Lengend Snippet: PBLD is a key target in cedrelone treated HCC. (A) Activation effect of cedrelone on PBLD expression in HepG2 and Hep3B cells was detected via western blotting after cells were treated with 20 µM cedrelone for 24 h. HepG2 and Hep3B cells were transfected with PBLD siRNA, which knocked down PBLD expression. HepG2 and Hep3B cells were treated with 20 µM cedrelone for 24 h. The effects of cedrelone on (B) PBLD expression, (C) cell growth, (D) cell viability and (E) cell death were detected in HepG2 and Hep3B cells via (B) western blotting, (C) sulforhodamine B staining, (D) a Cell Counting kit-8 assay and (E) trypan blue staining. HepG2 and Hep3B cells were transfected with PBLD siRNA and treated with 20 µM cedrelone for 24 h. (F) detection of cell invasion and (G) statistical analysis and (H) detection of cell migration and (I) statistical analysis. Scale bar, 200 µm for all eight images. HepG2 and Hep3B cells were transfected with PBLD siRNA or con-siRNA and treated with 20 µM cedrelone for 24 h. (J) epithelial-mesenchymal transition markers were detected through western blot. (K) The cell apoptosis were detected through FACScan flow cytometry, the abscissa represents Annexin V staining whilst the ordinate represents propidium iodide staining, and (L) statistical analysis. (M) The apoptosis markers were detected using western blot analysis. *P<0.05, **P<0.01 and ***P<0.005 as indicated. PBLD, phenazine biosynthesis-like domain-containing protein; siRNA, small interfering RNA; Con-, control; c-, cleaved; DMSO, dimethyl sulfoxide; PARP, poly-ADP ribose polymerase.

Article Snippet: HepG2 and Hep3B cells (1×10 4 ) were incubated with different concentrations (5, 10, 20 and 40 μM) of cedrelone (Shanghai Topscience Co., Ltd.) for 24 h at 37°C with 5% CO 2 and 10% trichloroacetic acid was added.

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Staining, Cell Counting, Migration, Flow Cytometry, Small Interfering RNA, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Novel compound cedrelone inhibits hepatocellular carcinoma progression via PBLD and Ras/Rap1

doi: 10.3892/etm.2019.8080

Figure Lengend Snippet: Ras and Rap1 pathway analysis in cedrelone-treated HepG2 cell. HepG2 cells were treated with 20 µM cedrelone for 24 h and analyzed using Illumina whole-genome expression arrays. GSEA analysis was performed after cedrelone treatment. Green represents the downregulation of genes and red represents the upregulation of genes. (A) Ras and (B) Rap1 signaling pathways were highlighted by pathway analysis. (A) A total of 16 genes were downregulated and 6 genes were upregulated in the Ras signaling pathway, and (B) 14 genes were downregulated in the Rap1 signaling pathway. HepG2 cells were transfected with PBLD siRNA to knockdown PBLD expression. The effect of cedrelone on genes of the (C) Ras and (D) Rap1 signaling pathways was significantly inhibited. (E) HepG2 and Hep3B cells were transfected with PBLD siRNA or con-siRNA and treated with 20 mM cedrelone for 24 h. Ras and Rap1 signaling pathway-associated molecules were detected via western blotting. Hep3B cells were transfected with PBLD siRNA and Ras/Rap1 overexpression vector or con-vector, and the (F) cell death ratio was detected using a trypan blue assay. (G) Cell viability was measured using a Cell Counting kit-8 assay. Additionally, (H) Hep3B cells were transfected with Ras or Rap1 overexpression vectors and the levels of Ras and Rap1, epithelial-mesenchymal transition phenotype markers (N-cadherin, E-cadherin and β-catenin) and the apoptosis marker c-capase-3 were detected via western blotting. *P<0.05, **P<0.01 and ***P<0.005 as indicated. GSEA, gene set enrichment analysis; Rap1, Ras-proximate-1; PBLD, phenazine biosynthesis-like domain-containing protein; siRNA, small interfering RNA; c-, cleaved; con-, control; DMSO, dimethyl sulfoxide; pERK, phosphorylated ERK.

Article Snippet: HepG2 and Hep3B cells (1×10 4 ) were incubated with different concentrations (5, 10, 20 and 40 μM) of cedrelone (Shanghai Topscience Co., Ltd.) for 24 h at 37°C with 5% CO 2 and 10% trichloroacetic acid was added.

Techniques: Expressing, Protein-Protein interactions, Transfection, Knockdown, Western Blot, Over Expression, Plasmid Preparation, Cell Counting, Marker, Small Interfering RNA, Control