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

Journal: The Journal of Neuroscience

Article Title: Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury

doi: 10.1523/JNEUROSCI.5267-14.2015

Figure Lengend Snippet: MET immunoreactivity was detected in a subset of myenteric plexus CGRP+ IPANs. A, MET immunoreactivity was detected in 34 ± 6% of HuC/D+ myenteric neurons of the adult mouse small bowel. All MET+ cells expressed the pan-neuronal marker HuC/D. B, MET staining was absent from S100B+ enteric glia. C, D, MET was detected in human myenteric neurons using colon cross sections. E, F, MET and RET are detected in mutually exclusive sets of neurons as confirmed by immunohistochemistry (E) and by using a RET-EGFP reporter mouse (F). G–I, 100% of MET+ neurons were CGRP+ and 50% of CGRP+ neurons were MET+. J–L, MET was also found in 16 ± 5% of calretinin+ cells and 8 ± 2% of MET+ cells are calretinin+. Arrows highlight a MET+ calretinin+ neuron. M–O, 12 ± 0.1% of ChAT+ cells were MET+ and 8 ± 0.1% of MET+ neurons were ChAT+. P–R, There was no overlap between MET and NADPH diaphorase-stained nitric oxide-producing neurons. S–U, HGF was detected in 43 ± 11% of MET+ neurons and 100% of the HGF+ neurons were MET+. V, In cross sections of E14.5 fetal bowel, HGF-IR was found in mesenchymal cells surrounding developing ENS stained with TuJ1 (W). X, At E14.5, MET+ cells were present in the region of developing ENS as well as in gut epithelium and mesenchymal cells. Scale bars in U applies to A, B, E–U. Scale bar in D applies to C and D. Scale bar in X applies to V–X. N ≥ 3 replicates/staining condition.

Article Snippet: Primary antibodies for mouse analysis were as follows: p75 NTR antibody (rabbit, 1:1000; #AB1554, EMD Millipore), Choline acetyltransferases (ChAT; goat, 1:10; #AB144P, Millipore), calretinin (rabbit, 1:2500; #AB5054, EMD Millipore), HGF (goat, 1:100; #sc-1357, Santa Cruz Biotechnology), HuC/D (mouse, 1:200; # {"type":"entrez-nucleotide","attrs":{"text":"A21272","term_id":"514140","term_text":"A21272"}} A21272 , Invitrogen), GFP (chicken, 1:1000; #GFP-1020, Aves Labs), S100B (rabbit, 1:800; DAKO), PGP9.5 (guinea pig, 1:100; #GP14104, Neuromics), TuJ1 (rabbit, 1:10,000; #PRB-435P, Covance), TuJ1 (mouse; #MMS-410P, 1:100, Covance), RET (goat, 1:800; #GT15002, 1:800, Neuromics), RET (R787) (Rabbit, 1:100; #18121, Immuno-Biological Laboratories), MET (goat, 1:100; AF527, R&D Systems), CGRP (rabbit, 1:100; #C8198, Sigma-Aldrich), phosphohistone 3 (pH3; rabbit, 1:800; #AB06-570, EMD Millipore), neuronal nitric oxide synthase (rabbit, 1:1000; AB#5380, EMD Millipore), substance P (rabbit, 1:1000; Inestar), vasoactive intestinal polypeptide (VIP; rabbit, 1:1000; Peninsula), NF145 (rabbit, 1:100; #AB1987, EMD Millipore).

Techniques: Marker, Staining, Immunohistochemistry

Journal: The Journal of Neuroscience

Article Title: Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury

doi: 10.1523/JNEUROSCI.5267-14.2015

Figure Lengend Snippet: HGF/MET signaling enhanced ENS precursor differentiation into neurons in vitro. A–H, E12.5 ENS precursors were maintained in culture after p75NTR immunoselection for 2 d in the presence or absence of 50 ng/ml HGF before immunohistochemistry using RET (A, B), TuJ1 (C, D), and pH3 (E, F) antibodies as well as DAPI nuclear staining (A–H). G, H, Merged images. I–K, While the total number of RET+ cells was not altered by HGF (I), the total number of TuJ1+ neurons (J) and the percentage of RET+ cells that were TuJ1+ (K) increased with HGF treatment. L, The number of dividing precursor cells (pH3 and RET double positive) decreased with HGF treatment, suggesting that HGF increased neuronal differentiation and decreased proliferation. White arrows, Nonmitotic RET+ pH3− ENS precursors. Yellow arrow, Mitotic RET+PH3+ ENS precursors. White arrowhead, RET+TuJ1+ neurons. Scale bar in G applies to all images (N = 3 biological replicates/group; 12 individual wells/group; *p < 0.01, Student's t test).

Article Snippet: Primary antibodies for mouse analysis were as follows: p75 NTR antibody (rabbit, 1:1000; #AB1554, EMD Millipore), Choline acetyltransferases (ChAT; goat, 1:10; #AB144P, Millipore), calretinin (rabbit, 1:2500; #AB5054, EMD Millipore), HGF (goat, 1:100; #sc-1357, Santa Cruz Biotechnology), HuC/D (mouse, 1:200; # {"type":"entrez-nucleotide","attrs":{"text":"A21272","term_id":"514140","term_text":"A21272"}} A21272 , Invitrogen), GFP (chicken, 1:1000; #GFP-1020, Aves Labs), S100B (rabbit, 1:800; DAKO), PGP9.5 (guinea pig, 1:100; #GP14104, Neuromics), TuJ1 (rabbit, 1:10,000; #PRB-435P, Covance), TuJ1 (mouse; #MMS-410P, 1:100, Covance), RET (goat, 1:800; #GT15002, 1:800, Neuromics), RET (R787) (Rabbit, 1:100; #18121, Immuno-Biological Laboratories), MET (goat, 1:100; AF527, R&D Systems), CGRP (rabbit, 1:100; #C8198, Sigma-Aldrich), phosphohistone 3 (pH3; rabbit, 1:800; #AB06-570, EMD Millipore), neuronal nitric oxide synthase (rabbit, 1:1000; AB#5380, EMD Millipore), substance P (rabbit, 1:1000; Inestar), vasoactive intestinal polypeptide (VIP; rabbit, 1:1000; Peninsula), NF145 (rabbit, 1:100; #AB1987, EMD Millipore).

Techniques: In Vitro, Immunohistochemistry, Staining

Journal: The Journal of Neuroscience

Article Title: Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury

doi: 10.1523/JNEUROSCI.5267-14.2015

Figure Lengend Snippet: HGF/MET signaling did not increase ENS precursor migration in culture. A–C, E12.5 gut slices were cultured 24 h on fibronectin-coated dishes with no added factor, HGF, or GDNF before staining for RET and TuJ1. A, ENCDCs migrate from the gut slice onto the culture dish even without any added factors. B, HGF did not increase the distance that ENCDCs migrated from the edge of the gut slice. C, GDNF markedly increased the distance ENCDCs migrate from the edge of the gut slice. D, Quantitative data (no added factor, 15 slices; 50 ng/ml HGF, 14 slices; 100 ng/ml HGF, 19 slices; 100 ng/ml GDNF, 30 slices; N = 3 independent experiments). *p < 0.001 for GDNF versus no added factor, ANOVA with Dunn's multiple-comparison test.

Article Snippet: Primary antibodies for mouse analysis were as follows: p75 NTR antibody (rabbit, 1:1000; #AB1554, EMD Millipore), Choline acetyltransferases (ChAT; goat, 1:10; #AB144P, Millipore), calretinin (rabbit, 1:2500; #AB5054, EMD Millipore), HGF (goat, 1:100; #sc-1357, Santa Cruz Biotechnology), HuC/D (mouse, 1:200; # {"type":"entrez-nucleotide","attrs":{"text":"A21272","term_id":"514140","term_text":"A21272"}} A21272 , Invitrogen), GFP (chicken, 1:1000; #GFP-1020, Aves Labs), S100B (rabbit, 1:800; DAKO), PGP9.5 (guinea pig, 1:100; #GP14104, Neuromics), TuJ1 (rabbit, 1:10,000; #PRB-435P, Covance), TuJ1 (mouse; #MMS-410P, 1:100, Covance), RET (goat, 1:800; #GT15002, 1:800, Neuromics), RET (R787) (Rabbit, 1:100; #18121, Immuno-Biological Laboratories), MET (goat, 1:100; AF527, R&D Systems), CGRP (rabbit, 1:100; #C8198, Sigma-Aldrich), phosphohistone 3 (pH3; rabbit, 1:800; #AB06-570, EMD Millipore), neuronal nitric oxide synthase (rabbit, 1:1000; AB#5380, EMD Millipore), substance P (rabbit, 1:1000; Inestar), vasoactive intestinal polypeptide (VIP; rabbit, 1:1000; Peninsula), NF145 (rabbit, 1:100; #AB1987, EMD Millipore).

Techniques: Migration, Cell Culture, Staining