Journal: PLoS ONE

Article Title: Tocilizumab does not block interleukin-6 (IL-6) signaling in murine cells

doi: 10.1371/journal.pone.0232612

Figure Lengend Snippet: (A) Human U-937 cells were pretreated with tocilizumab as indicated for 30 min and then stimulated with 10 ng/ml hIL-6 for 15 min. Phosphorylation of STAT3 was determined by western blot. Quantification of three independent experiments and one representative western blot are shown. (B-D) The experiments were performed as described under (A) but with murine RAW264.7 cells and mIL-6 (B), with human HepG2 cells and hIL-6 (C) and with murine AML-12 cells and mIL-6 (D). Statistical significance was analyzed with one-way ANOVA followed by Dunnett's multiple comparisons test (**: p<0.01; ***: p < 0.001; ns: not significant).

Article Snippet: The human monocyte cell line U-937 (CRL-1593.2) and the murine macrophage cell line RAW264.7 (SC-6003) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the human hepatocyte cell line HepG2 (ACC 180) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Phospho-proteomics, Western Blot

Journal: PLoS ONE

Article Title: Khellin and Visnagin Differentially Modulate AHR Signaling and Downstream CYP1A Activity in Human Liver Cells

doi: 10.1371/journal.pone.0074917

Figure Lengend Snippet: A) HepG2 cells were treated with 10 µM visnagin, 10 µM khellin, and/or DMSO for 4 h, 8 h, 16 h, and 24 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. * - value is significantly different from DMSO-treated cells ( p < 0.05). B) HepG2 cells were pre-treated with 20 µM MNF for 1 h and then exposed to 10 µM visnagin or 10 µM khellin for additional 16 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. * - value is significantly different from DMSO-treated cells ( p < 0.05). # - value is significantly reduced in comparison to cells treated with VIS and KHEL, respectively; ( p < 0.05). C) HepG2 cells were treated with visnagin (VIS; 1 µM-20 µM), khellin (KHEL; 1 µM-20 µM), 1 µM 3MC, 5 nM TCDD, and/or vehicle (DMSO; 0.1% v/v) for 48 h. Thereafter, western blotting analyses for detection of CYP1A1 and actin were performed as described in Materials and Methods section. The representative western blot analysis of two independent experiments (passages) is presented. D) HepG2 cells were treated with visnagin (VIS; 1 µM-20 µM), khellin (KHEL; 1 µM-20 µM), 1 µM 3MC, 5 nM TCDD, and/or vehicle (DMSO; 0.1% v/v) for either 16 h (upper panel) or 48 h (lower panel). EROD activity was determined as described in Materials and Method section. Analyses were performed in three independent experiments and are shown as fold induction over untreated cells. * - value is significantly different from DMSO-treated cells ( p < 0.05). E) HepG2 cells were treated with TCDD (5 nM) for 48 h. Thereafter, substrate mixture was supplemented with increasing doses of visnagin (VIS 1 nM -20 µM) or khellin (KHEL; 1 nM -20 µM) and EROD activity was determined as described in Materials and Methods section. Data are mean from three independent experiments and are expressed as percentage (%) of TCDD-mediated induction (i.e. induction by TCDD = 100%). * - value is significantly different from TCDD-treated cells ( p < 0.005).

Article Snippet: Blots were probed with primary antibodies against CYP1A1 (goat polyclonal, sc-9828, G-18, diluted 1:500 – for detection in human hepatocytes; rabbit polyclonal, sc-20772, H-70, diluted 1:500 – for detection in HepG2 cells), CYP1B1 (mouse monoclonal, sc-374228, G-4, 1:1000), actin (goat polyclonal; sc-1616, 1-19, diluted 1:2000), all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or GAPDH (rabbit monoclonal, 2118, 14C10, diluted 1:1000) purchased from Cell Signaling Technology, overnight at 4°C.

Techniques: Control, Comparison, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Khellin and Visnagin Differentially Modulate AHR Signaling and Downstream CYP1A Activity in Human Liver Cells

doi: 10.1371/journal.pone.0074917

Figure Lengend Snippet: Influence of visnagin and khellin treatment on CYP1A1 mRNA expression in human primary hepatocytes.

Article Snippet: Blots were probed with primary antibodies against CYP1A1 (goat polyclonal, sc-9828, G-18, diluted 1:500 – for detection in human hepatocytes; rabbit polyclonal, sc-20772, H-70, diluted 1:500 – for detection in HepG2 cells), CYP1B1 (mouse monoclonal, sc-374228, G-4, 1:1000), actin (goat polyclonal; sc-1616, 1-19, diluted 1:2000), all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or GAPDH (rabbit monoclonal, 2118, 14C10, diluted 1:1000) purchased from Cell Signaling Technology, overnight at 4°C.

Techniques: Expressing