Journal: American Journal of Cancer Research

Article Title: Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori -activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

doi:

Figure Lengend Snippet: Hp-hAGF secretome induces EMT related changes of motility mode in cancer cells. Nomarski contrast of the phenotype of HT29 cells placed in DMEM+10% FBS (control), hGF supernatant and Hp-hAGF supernatant (A) and of AGS cells placed in DMEM/F12HAM+10% FBS, hGF supernatant, Hp-hAGF supernatant and Hp-hAGF supernatant with the addition of EGFR inhibitor tyrphostin A46 (0.1 mM) (B). Cell trajectories of epithelioid AGS cells in DMEM/F12HAM+10% FBS, hGF supernatant, Hp-hAGF supernatant and Hp-hAGF supernatant with the addition of A46 (0.1 mM) (C) and corresponding motility parameters (D). Cell trajectories of fibroblastoid AGS cells in DMEM/F12HAM+10% FBS, hGF supernatant, Hp-hAGF supernatant and Hp-hAGF supernatant with the addition of A46 (0.1 mM) and corresponding motility parameters (E). Cell trajectories are presented as circular diagrams (axis scale in µm) drawn with the initial point of each trajectory placed at the origin of the plot. Total length of cell trajectory (µm), velocity of cell movement (speed; total length of cell trajectory/time of recording; µm/min) and total length of cell displacement (i.e. the distance from the starting point directly to the cell final position; µm) were quantified with the Hiro program. Column charts show migration parameters at the population level (registered for 8 h; N=50): distance and displacement.

Article Snippet: For the experiments with AGS cells (ATCC CRL-1739, Manassas, VA, USA), the supernatant from Hp -activated human gastric fibroblasts ( Hp -hAGF) and from normal human gastric fibroblasts (hGF) was collected as described previously [ 19 , 28 ].

Techniques: Control, Migration

Journal: American Journal of Cancer Research

Article Title: Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori -activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

doi:

Figure Lengend Snippet: Hp-hAGF secretome prompts Twist related EMT type 3 pro-pluripotent changes in cancer cells. RT-PCR analysis of the expression of mRNA for 18S, Snail, Twist and pluripotency-related Yamanaka factors: c-Myc, Oct4 and Sox-2 in HT29 (A) and AGS (B) cells cultured for 96 hrs in control medium, hGF and Hp-hAGF supernatants and the ratio of selected genes over 18S mRNA. Additionally AGS cells were cultured in Hp-hAGF secretome with the addition of A46 (0.1 mM). The analysis shows Snail mRNA expression increase and strong Twist transcriptional upregulation. Results are mean ± SEM of four independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control value. Hash (#) indicates a significant change (P<0.05) as compared to the value for cells cultured in hGF secretome.

Article Snippet: For the experiments with AGS cells (ATCC CRL-1739, Manassas, VA, USA), the supernatant from Hp -activated human gastric fibroblasts ( Hp -hAGF) and from normal human gastric fibroblasts (hGF) was collected as described previously [ 19 , 28 ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Control

Journal: American Journal of Cancer Research

Article Title: Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori -activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype

doi:

Figure Lengend Snippet: Hp-hAGF secretome induces increased invasiveness of cancer cells. This phenomenon in adenocarcinoma cells is related to cMet/HGF/Integrin-β1/TNC and EGFR signaling. Geltrex invasion assay of HT29 and AGS cells towards culture medium, hGF and Hp-hAGF supernatants and in the case of AGS cells towards Hp-hAGF secretome with the addition of tyrphostin A46 (0.1 mM) showing enhanced invasiveness towards Hp-hAGF supernatant and the decrease after A46 (A). Fluorescent microscopy of transmigrated AGS cells attached to the bottom side of the geltrex/transwell membrane stained with bisbenzimide (Hoechst 33342) (B). The phase contrast microscopy showing HT29 and AGS cells that transmigrated through invasion chambers towards Hp-hAGF supernatant. Three independent experiments were performed for each condition (C). RT-PCR analysis of the expression of 18S, Fap, MMP 2, 3, 9 and mRNA in HT29 (D) and AGS (E) cells cultured in culture medium, hGF, Hp-hAGF supernatants and in the case of AGS cells in Hp-hAGF secretome with addition of tyrphostin A46 (0.1 mM) and the ratio of selected genes over 18S mRNA showing their strong upregulation in Hp-hAGF secretome. Phase contrast microscopy of Geltrex metalloproteinase activity assay (1:1) of HT29 (F) and AGS (G) flooded with culture medium, hGF, Hp-hAGF supernatants and in the case of AGS cells with Hp-hAGF secretome with the addition of A46 (0.1 mM), showing enhanced ability of cells flooded with Hp-hAGF supernatant to cross basement membrane components already after 24 hrs. RT-PCR analysis of the mRNA expression for 18S, cMet, HGF, EGFR and Integrin-β1 in AGS cells cultured in control medium, hGF, Hp-hAGF and in Hp-hAGF secretome with the addition of A46 (0.1 mM) and the ratio of selected genes over 18S mRNA (H). Results are mean ± SEM of four independent experimental repeats. Asterisk (*) indicates a significant change (P<0.05) as compared to the control cell value. Hash (#) indicates a significant change (P<0.05) as compared to the value of cells grown in GF supernatant.

Article Snippet: For the experiments with AGS cells (ATCC CRL-1739, Manassas, VA, USA), the supernatant from Hp -activated human gastric fibroblasts ( Hp -hAGF) and from normal human gastric fibroblasts (hGF) was collected as described previously [ 19 , 28 ].

Techniques: Invasion Assay, Microscopy, Membrane, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Activity Assay, Control

Journal: Clinical Science (London, England : 1979)

Article Title: MIB1 mutations reduce Notch signaling activation and contribute to congenital heart disease

doi: 10.1042/CS20180732

Figure Lengend Snippet: HEK 293T cells were co-transfected with Flag-tagged full-length JAG1 (JAG1-Flag), tGFP-tagged full-length MIB1 wild-type or mutants (MIB1-tGFP) and HA-tagged tetrameric ubiquitin (HA-4 Ub). Cells were pre-treated with 10 mM MG132 for 6 h before harvest at 24 h post-transfection. Immunoprecipitation was performed with Flag antibody coupled beads, and samples were detected with respective antibodies. Western blot by HA antibody reveals ubiquitination level of JAG1.

Article Snippet: Myc-Flag-tagged full-length human JAG1 (pCMV6-Entry-JAG1) was purchased from Origene (#RC210516). pCMV6-AN-DDK-JICD was generated using JAG1 intracellular domain (aa 1092-1218) subcloned into SgfI/MluI sites of pCMV6-AN-DDK (Origene, #PS100014).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot

Journal: Clinical Science (London, England : 1979)

Article Title: MIB1 mutations reduce Notch signaling activation and contribute to congenital heart disease

doi: 10.1042/CS20180732

Figure Lengend Snippet: HEK 293T cells were co-transfected with Flag-tagged JAG1 intracellular domain (Flag-JICD) and HA-tagged full-length MIB1 wild-type or mutants (HA-MIB1) ( A , statistic assay of band density was calculated from two independent experiments) or Myc-tagged mib repeats domain region of MIB1 wild-type or variants (Myc-mib reps) ( B ). Twenty-four hours post-transfection, cell lysate was immunoprecipitated with Flag antibody immobilized beads, and samples were detected with respective antibodies.

Article Snippet: Myc-Flag-tagged full-length human JAG1 (pCMV6-Entry-JAG1) was purchased from Origene (#RC210516). pCMV6-AN-DDK-JICD was generated using JAG1 intracellular domain (aa 1092-1218) subcloned into SgfI/MluI sites of pCMV6-AN-DDK (Origene, #PS100014).

Techniques: Transfection, Immunoprecipitation

Journal: Journal of Diabetes Research

Article Title: The Involvement of Notch1-RBP-Jk/Msx2 Signaling Pathway in Aortic Calcification of Diabetic Nephropathy Rats

doi: 10.1155/2017/8968523

Figure Lengend Snippet: The mRNA expressions of Notch1 (a), RBP-Jk (b), Msx2 (c), and Jagged1 (d) in aortic tissues. Data presented as mean ± standard deviation (SD). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus Nor group; Δ p > 0.05, # p < 0.05, ## p < 0.01, and ### p < 0.001. Nor group: normal controls; DN + VDN group: diabetic nephropathy rats with vitamin D3/nicotine-induced vascular calcification.

Article Snippet: The following primary antibodies were used for immunohistochemical analysis: monoclonal rabbit anti-rat RBP-Jk, Msx2, and N1-ICD (Shanghai Bioworld Tech Inc., China); monoclonal rabbit anti-rat Jagged1 and Runx2 (Beijing BioTech Inc., China); monoclonal rabbit anti-rat Notch1 (Wuhan Proteintech Group Inc., China); and monoclonal mouse anti-rat α -SMA (Wuhan Boster Bio-Tech., Ltd, China).

Techniques: Standard Deviation

Journal: Journal of Diabetes Research

Article Title: The Involvement of Notch1-RBP-Jk/Msx2 Signaling Pathway in Aortic Calcification of Diabetic Nephropathy Rats

doi: 10.1155/2017/8968523

Figure Lengend Snippet: Detection of Notch1, RBP-Jk, Msx2, Jagged1, and N1-ICD levels by immunohistochemical analysis. The representative images and quantitative analysis of Notch1 (a-b), RBP-Jk (c-d), Msx2 (e-f), Jagged1 (g-h), and N1-ICD (i-j) in aortic tissues. The left panel of histological images: Nor group; right panel: DN + VDN group at 8, 12 , and 16 weeks, respectively. Data presented as mean ± standard deviation (SD). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus Nor group; Δ p > 0.05, # p < 0.05, ## p < 0.01, and ### p < 0.001. Nor group: normal controls; DN + VDN group: diabetic nephropathy rats with vitamin D3/nicotine-induced vascular calcification.

Article Snippet: The following primary antibodies were used for immunohistochemical analysis: monoclonal rabbit anti-rat RBP-Jk, Msx2, and N1-ICD (Shanghai Bioworld Tech Inc., China); monoclonal rabbit anti-rat Jagged1 and Runx2 (Beijing BioTech Inc., China); monoclonal rabbit anti-rat Notch1 (Wuhan Proteintech Group Inc., China); and monoclonal mouse anti-rat α -SMA (Wuhan Boster Bio-Tech., Ltd, China).

Techniques: Immunohistochemical staining, Standard Deviation

Journal: Neurobiology of aging

Article Title: Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease.

doi: 10.1016/j.neurobiolaging.2015.01.001

Figure Lengend Snippet: Fig. 1. Prostate stem cell antigen (PSCA) is soluble and co-purifies with the a4 nAChR subunit in human cortex. (A) The antiserum directed against PSCA recognizes the human recombinant GST-tagged PSCA protein (total molecular weight 34.2 kDa) from 1 to 16 ng/well at the expected band size. Endogenous PSCA from mouse and human cortical tissue (8 mg total protein/well) is detected at approximately 24 kDa, and the amount in the cortical samples was compared with the known concentrations of re- combinant PSCA protein. (B) Images of Western blot showing PSCA protein levels in cortical tissue from human (protein concentration 4, 8, and 12 mg/well) and mouse (8, 15, and 30 mg/well) in the absence (peptide) and the presence of PSCA peptide (þpeptide). (C) Representative images of Western blots showing PSCA and Lypd6 protein levels in soluble and membrane fractions of human temporal cortical tissue. The membrane receptor proteins b2 nAChR and GluR2 are used as control. (D) Mag- netic beads covalently coupled with PSCA recombinant protein were incubated with cortical homogenates from human temporal cortex followed by detection of nAChR subunits by Western blot. Homogenates before (input) and after affinity purification (output) as well as the negative control (CTRL) were loaded.

Article Snippet: Validation of PSCA antibody (1:1,000, Novus Biologicals) was done with co-incubation with the PSCA peptide, which had been used as immunogen (1:100, # NB100-91938PEP, Novus Biologicals).

Techniques: Recombinant, Molecular Weight, Western Blot, Protein Concentration, Membrane, Control, Incubation, Negative Control