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: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Effect of adipose-derived mesenchymal stem cells and their conditioned media on hepatocellular carcinoma cell proliferation and apoptosis. HCC cells (2 × 10 5 ) were seeded in six-well coculture plates in the presence or absence of ADMSCs and ADMSC CM, undiluted or diluted 1:5 or 1:25 for 48 h. The proliferation of HepG2 and PLC-PRF-5 HCC cell lines were measured by (A) cell count assay and (B) WST-8 proliferation tests; The apoptosis of HepG2 (C) and PLC-PRF (D) cells co-cultured as above was measured by flow cytometry using Annexin V/PI test kit; C and D: Two representative experiments of apoptosis in HepG2 and PLC-PRF cells, respectively; E: The average rate of apoptosis in HepG2 and PLC-PRF-5 cells induced by ADMSCs, ADMSC CM. Data are representative of three independent experiments, each repeated in triplicate. All data are represented as mean ± SD ( a P < 0.05, b P < 0.01, c P < 0.001). CTR: Control; ADMSC: Adipose-derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The HepG2/C3A and PLC/PRF/5 cell migration assay was performed using a Boyden chamber in a 24-well plate designed by Cell Biolabs Inc. (San Diego, CA, United States) according to the manufacturer's recommendations.

Techniques: Derivative Assay, Cell Counting, Cell Culture, Flow Cytometry

Journal: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Effect of adipose derived mesenchymal stem cells and their conditioned media on of hepatocellular carcinoma cell migration and invasion. HCC cells (2 × 10 5 ) were seeded in six- well co-culture plates in the presence or absence of ADMSCs (at ADSMCs: HCC ratio of 1:1 or 1:2) or ADMSC CM, undiluted or diluted 1:5 or 1:25. The migration of (A) HepG2 and (B) PLC-PRF-5 cells was assessed by wound healing assay. The migration rate at 24 h is represented in (C); D: A Transwell migration assay was performed to confirm the results of the wound healing assay; E: HCC cell invasiveness was measured by Transwell invasion assay. In the Transwell migration and invasion assay, 3 × 10 5 HCC cells alone, co-cultured with ADMSCs, or treated with ADMSC CM were seeded into the apical chamber of Transwell plates and allowed to migrate or invade through the uncoated polycarbonate membrane or collagen-coated polycarbonate membrane, respectively (8 μm pore size) to the lower chamber for 24 h or 48 h, respectively. The migratory or invasive cells were stained with crystal violet cell stain solution and extracted using an extraction solution provided in the kit. The level of migration and invasion was measured using a plate reader at the absorbance of 560 nm. Values shown are representative of five independent experiments, each performed in triplicate. Data are represented as mean ± SD of five independent experiments, each performed in triplicate ( a P < 0.05, b P < 0.01, c P < 0.001). CTR: Control; ADMSC: Adipose derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The HepG2/C3A and PLC/PRF/5 cell migration assay was performed using a Boyden chamber in a 24-well plate designed by Cell Biolabs Inc. (San Diego, CA, United States) according to the manufacturer's recommendations.

Techniques: Derivative Assay, Migration, Co-Culture Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay, Invasion Assay, Cell Culture, Staining

Journal: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Effect of adipose-derived mesenchymal stem cells and adipose-derived mesenchymal stem cell conditioned media on tissue inhibitor metalloproteinase mRNA levels in hepatocellular carcinoma cells. HCC cells (2 × 10 5 ) were seeded in six-well co-culture plates in the presence or absence of ADMSCs or of undiluted ADMSC conditioned media (CM). The mRNA levels of TIMP-1, TIMP-2, and TIMP-3 in HepG2 (A) and PLC-PRF-5 (B) cells after the removal of ADMSCs in the case of coculture was measured by quantitative PCR. Data are represented as mean ± SD of five independent experiments, each performed in triplicate ( a P < 0.05, b P < 0.01, c P < 0.001). ADMSC: Adipose-derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The HepG2/C3A and PLC/PRF/5 cell migration assay was performed using a Boyden chamber in a 24-well plate designed by Cell Biolabs Inc. (San Diego, CA, United States) according to the manufacturer's recommendations.

Techniques: Derivative Assay, Co-Culture Assay, Real-time Polymerase Chain Reaction

Journal: World Journal of Gastroenterology

Article Title: Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

doi: 10.3748/wjg.v25.i5.567

Figure Lengend Snippet: Expression of tumor suppressor genes and oncogenes in hepatocellular carcinoma cells. HCC cells (2 × 10 5 ) were seeded in six-well co-culture plates in the presence or absence of ADMSCs or undiluted ADMSC CM for 48 h. The mRNA expression of the tumor suppressor genes P53/RB, oncogene c-Myc and the enzymatic component of telomerase hTERT were assessed by RT-PCR in (A) HepG2 and (B) PLC-PRF-5 cells after the removal of ADMSCs in the case of co-culture. Data are represented as mean ± SD of five independent experiments, each performed in triplicate ( a P < 0.05, b P < 0.01, c P < 0.001). ADMSC: Adipose-derived mesenchymal stem cell; CM: Conditioned media; HCC: Hepatocellular carcinoma.

Article Snippet: The HepG2/C3A and PLC/PRF/5 cell migration assay was performed using a Boyden chamber in a 24-well plate designed by Cell Biolabs Inc. (San Diego, CA, United States) according to the manufacturer's recommendations.

Techniques: Expressing, Co-Culture Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay