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: Cellular and Molecular Gastroenterology and Hepatology

Article Title: STAT3 is Activated by CTGF-mediated Tumor-stroma Cross Talk to Promote HCC Progression

doi: 10.1016/j.jcmgh.2022.09.006

Figure Lengend Snippet: Stromal cells increased p-STAT3 and CTGF expression and cell growth in hepatoma cells. HepG2 or Alex cells were transfected with STAT3 siRNA or control siRNA ( A-B ). WST-8 assay ( A ) and CTGF mRNA expression ( B ) of HepG2 or Alex cells after siRNA transfection. Total RNA was collected 48 hours after siRNA transfection. HepG2 or Alex cells were incubated in monoculture or cocultured with LX-2, THP-1, MTA, and TMNK-1 cells, using Transwell insert system ( C-G ). THP-1 cells were pretreated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce macrophage differentiation before coculturing. Total proteins and RNA were collected after 24 hours of incubation ( C, E, F ). C , Western blot showing protein expression of HepG2 or Alex cells. D , WST-8 assay of HepG2 or Alex cells in coculture at the indicated time points. HepG2 or Alex cells were transfected with STAT3 siRNA, gp130 siRNA, or control siRNA 48 hours before coculturing ( E-G ). E , Western blot showing protein expression and qPCR showing CTGF mRNA expression in HepG2 or Alex cells transfected with STAT3 siRNA or control siRNA. F , Western blot and CTGF mRNA expression in HepG2 or Alex cells transfected with gp130 siRNA or control siRNA. G , WST-8 assay of HepG2 or Alex cells transfected with STAT3 siRNA, gp130 siRNA, or control siRNA 48 hours after incubation (n = 3-4). ∗ , †, ‡, § P < .05 vs control.

Article Snippet: The HepG2 and Alex human hepatoma cell lines were obtained from the Japanese Collection of Research Bioresources/Health Science Research Resources Bank (JCRB/HSRRB) cell bank (Osaka, Japan) (JCRB Cat# NIHS0326, RRID:CVCL 0027 and JCRB Cat# JCRB0406, RRID:CVCL_0485, respectively).

Techniques: Expressing, Transfection, Control, Incubation, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: STAT3 is Activated by CTGF-mediated Tumor-stroma Cross Talk to Promote HCC Progression

doi: 10.1016/j.jcmgh.2022.09.006

Figure Lengend Snippet: IL-6 family cytokines increased p-STAT3 and CTGF expression in hepatoma cells and enhanced cell proliferation. Concentrations of IL-6, LIF, and OSM in the culture supernatant ( A ). Culture supernatant was collected 24 hours after the initiation of HepG2, Alex, LX-2, THP-1, MTA, and TMNK-1 cell monoculture. IL-6, LIF, or OSM concentration was measured by enzyme-linked immunosorbent assay. THP-1 cells were pretreated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce macrophage differentiation, and the culture medium was replaced with PMA-free medium 24 hours before supernatant collection. HepG2 cells and Alex cells were treated with recombinant proteins of IL-6 (20 ng/mL), LIF (1:1000), or OSM (20 ng/mL) ( B-D ). Cells were serum-starved for 16 hours before the addition of recombinant proteins. B , Protein expression 1 hour after recombinant protein treatment. C , mRNA expression of CTGF 3 hours after recombinant protein treatment. D , WST-8 assay at the indicated time points after treatment with recombinant proteins. HepG2 cells and Alex cells were transfected with gp130 siRNA, STAT3 siRNA, or control siRNA 48 hours before the addition of recombinant proteins ( E-F ). Recombinant proteins were added 16 hours after serum starvation. E , mRNA expression of CTGF 3 hours after treatment with recombinant proteins. F , WST-8 assay at the indicated time points after recombinant protein treatment (n = 3-4). ∗ P < .05.

Article Snippet: The HepG2 and Alex human hepatoma cell lines were obtained from the Japanese Collection of Research Bioresources/Health Science Research Resources Bank (JCRB/HSRRB) cell bank (Osaka, Japan) (JCRB Cat# NIHS0326, RRID:CVCL 0027 and JCRB Cat# JCRB0406, RRID:CVCL_0485, respectively).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Transfection, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: STAT3 is Activated by CTGF-mediated Tumor-stroma Cross Talk to Promote HCC Progression

doi: 10.1016/j.jcmgh.2022.09.006

Figure Lengend Snippet: Knockdown of CTGF in HepG2 cells decreased IL-6 family cytokine expression in stromal cells, downregulated p-STAT3 expression in HepG2 cells and reduced the proliferation of HepG2 cells. Western blotting of HepG2 or Alex cells treated with recombinant CTGF protein under monoculture ( A ). Protein lysate was collected from cells 0, 0.5, 1, and 3 hours after the addition of 5 nM recombinant CTGF protein. HepG2 cells were incubated under monoculture or coculture with stromal cell lines ( B-E ). HepG2 cells were transfected with CTGF siRNA or control siRNA 48 hours before coculturing. Total proteins and RNA were collected after 24 hours of coculture. THP-1 cells had been pretreated with 100 ng/m phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce macrophage differentiation before coculture. B , Western blot showing HepG2 cell proteins in monoculture or cocultured with LX-2, THP-1, MTA, and TMNK-1 cells. C , WST-8 assay of HepG2 cells performed 48 hours after initiation of monoculture or coculture (n = 3). ∗ P < .05. D , mRNA expression of IL-6 family cytokines in stromal cell lines in monoculture or cocultured with HepG2 cells transfected with CTGF siRNA or control siRNA (n = 4). E , IL-6 concentration in supernatant measured via enzyme-linked immunosorbent assay (n = 4). ∗ P < .05 vs monoculture; ∗∗ P < .05 vs coculture with HepG2 control si.

Article Snippet: The HepG2 and Alex human hepatoma cell lines were obtained from the Japanese Collection of Research Bioresources/Health Science Research Resources Bank (JCRB/HSRRB) cell bank (Osaka, Japan) (JCRB Cat# NIHS0326, RRID:CVCL 0027 and JCRB Cat# JCRB0406, RRID:CVCL_0485, respectively).

Techniques: Knockdown, Expressing, Western Blot, Recombinant, Incubation, Transfection, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay