Journal:

Article Title: Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

doi: 10.1128/IAI.73.8.4643-4652.2005

Figure Lengend Snippet: Cell lines used for this study

Article Snippet: Cells were grown in the indicated media and subcultured every 2 to 3 days. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Cell line Reference or source a (no.) Cell type Growth medium b 293T ATCC (CRL 11268) Human embryonal kidney DMEM (Gibco BRL), 10% fetal bovine serum (FBS; Gibco BRL), 2 mM l -glutamine, 1 mM sodium pyruvate AGS ATCC (CRL 1739) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine CHO K1 ATCC (CCL 61) Chinese hamster ovary MEM alpha (Gibco BRL), 10% FBS Cos1 ATCC (CRL 1650) Green African monkey kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine GLC4 42 Human lung carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Hec1.b ATCC (HTP 113) Human endometrial adenocarcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine Hela ATCC (CCL 2) Human cervix epithelial carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HepG2 ATCC (HB 8065) Human hepatocellular carcinoma RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HL ECACC (96121720) Human lung, fibroblast-like RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine HT29 ATCC (HTB 38) Human colon adenocarcinoma Mc Coy's 5A (Gibco BRL), 10% FBS J774.A DSMZ (ACC 170) Mouse monocytic macrophages RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Kato3 ATCC (HTB 103) Human gastic carcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine L929 ATCC (CCL 1) Mouse fibroblasts RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MDCK ATCC (CCL 34) Canine kidney RPMI 1640 (Gibco BRL), 10% FBS, 2 mM l -glutamine MKN-28 27 Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine MKN-45 DSMZ (ACC 409) Human gastric adenocarcinoma RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine SR4987 DSMZ (ACC 323) Mouse stromal fibroblast-like Mc Coy's 5A (Gibco BRL), 10% FBS SYF+src ATCC (CRL 2498) Mouse fibroblasts overexpressing Src DMEM (Gibco BRL), 10% FBS, 2 mM l -glutamine, 1 mM sodium pyruvate THP1 ATCC (TIB 202) Human monocytes from acute monocytic leukemia RPMI 1640 (Gibco BRL), 10% heat-inactivated FBS, 2 mM l -glutamine Open in a separate window a ATCC, American Type Culture Collection ( www.atcc.org ); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ( www.dsmz.de ); ECACC, European Collection of Cell Cultures ( www.ecacc.org.uk ). b DMEM, Dulbecco's modified Eagle's medium; MEM, minimum essential medium.

Techniques:

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 3. Microscopic image and growth curve of HEK293 WT and RH2A-KO cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques:

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 5. Expression of RH2A.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Expressing

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 6. Expression of RH2A variants with AGS- causing mutation in RH2A-KO cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Expressing, Mutagenesis

Journal: Cellular microbiology

Article Title: Dual infection system identifies a crucial role for PKA-mediated serine phosphorylation of the EPEC-Tir-injected effector protein in regulating Rac1 function.

doi: 10.1111/j.1462-5822.2009.01330.x

Figure Lengend Snippet: Fig. 2. Intimin and Tir of EPEC play a role in blocking H. pylori-induced actin-cytoskeletal rearrangements. A. AGS cells were infected with H. pylori in the presence or absence of wt and isogenic mutant EPEC strains as indicated. Inhibition of the H. pylori-induced host cell elongation was evident with wt EPEC but not EPECDescN or EPECDespB mutants that lack a functional T3SS, suggesting that one or more T3SS effector proteins are essential for the blocking activity. To identify the EPEC factor(s) involved, co-infection experiments with Dmap, DespF, Dtir and Deae mutants were performed. Only the Dtir and Deae mutants abolished the blocking effect, suggesting that either Tir–Intimin interaction is critical for EPEC to block the H. pylori-induced phenotypic response. B. Tyrosine phosphorylation of CagA during co-infection was analysed by Western blotting using an a-phosphotyrosine (a-P-Tyr) antibody and an a-CagA antibody. Expression and size shifting of Tir during co-infection was analysed by Western blotting using an a-Tir antibody. Arrows indicate the Tir forms on the gel. The size shift of Tir (asterisk) was reported to be dependent on PKA-mediated serine phosphorylation of Tir rather than its tyrosine phosphorylation (Warawa and Kenny, 2001). C. Genetic complementation of Intimin and Tir restore the ability of EPEC mutants to block H. pylori-induced actin-cytoskeletal rearrangements. AGS cells were infected with H. pylori in the presence or absence of complemented EPECDtir/tir or EPECDeae/eae strains which restore the function of the genes. Western blots were done as shown in B. D. The number of elongated AGS cells in each experiment was quantified in 10 different 0.25 mm2 fields. The capacity to induce elongation of infected AGS cells is represented as efficient induction (60–75% or + phenotype), drastically reduced or no induction (< 10%, or – phenotype). Infections were carried out for 4 h using moi = 100 (H. pylori) or moi = 40 (EPEC) (B and C). The results are representative from at least three independent experiments.

Article Snippet: Cell culture, transient transfection and infection assays AGS cells (ATCC CRL 1739a human gastric adenocarcinoma epithelial cell line) were grown in 6 well plates containing RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) supplemented with 25 mM Hepes buffer and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany) for 2 days to reach monolayers of approximately 70% cell confluence.

Techniques: Blocking Assay, Infection, Mutagenesis, Inhibition, Functional Assay, Activity Assay, Phospho-proteomics, Western Blot, Expressing

Journal: Cellular microbiology

Article Title: Dual infection system identifies a crucial role for PKA-mediated serine phosphorylation of the EPEC-Tir-injected effector protein in regulating Rac1 function.

doi: 10.1111/j.1462-5822.2009.01330.x

Figure Lengend Snippet: Fig. 3. Distinct effects of Tir deletion mutants on H. pylori-induced actin-cytoskeletal rearrangements. A. Schematic representation of EPEC and EHEC Tir constructs used in this experiment (Kenny, 2001). Numbers indicate the amino acid residue positions corresponding to the deletions made in Tir. The two transmembrane domains (TM) flanking the Intimin-binding domain (IBD) and the two known tyrosine phosphorylation sites (Y454 and Y474) in EPEC Tir are indicated. B. AGS cells were infected with H. pylori in the presence or absence of wt and isogenic EPEC mutant strains expressing these Tir variants in either EPECDtir or EPECDtir/eae double mutant. Expression of wt Tir in the tir mutant restored the strain’s ability to inhibit H. pylori-induced elongation. Interestingly, deletion of amino acid residues 77–277, 277–478 or 77–478 abolished Tir-inhibitory effect, while Tir deleted for residues 183–332 retained intermediate inhibitory activity. The number of elongated AGS cells in each co-infection experiment was quantified in 10 different 0.25 mm2 fields. Infections were carried out for 4 h using moi = 100 (H. pylori) or moi = 40 (EPEC). The results are representative from at least three independent experiments. P-values of <0.005 (**) and <0.05 (*) were considered as significant. C. Similar expression of the EPEC Tir constructs was analysed by Western blotting using an a-Tir antibody. Arrows indicate the different Tir variants on the gel. D. As control, tyrosine phosphorylation of CagA during co-infection was also analysed by Western blotting using a-phosphotyrosine and a-CagA antibodies.

Article Snippet: Cell culture, transient transfection and infection assays AGS cells (ATCC CRL 1739a human gastric adenocarcinoma epithelial cell line) were grown in 6 well plates containing RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) supplemented with 25 mM Hepes buffer and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany) for 2 days to reach monolayers of approximately 70% cell confluence.

Techniques: Construct, Residue, Binding Assay, Phospho-proteomics, Infection, Mutagenesis, Expressing, Activity Assay, Western Blot, Control

Journal: Cellular microbiology

Article Title: Dual infection system identifies a crucial role for PKA-mediated serine phosphorylation of the EPEC-Tir-injected effector protein in regulating Rac1 function.

doi: 10.1111/j.1462-5822.2009.01330.x

Figure Lengend Snippet: Fig. 4. The tyrosine phosphorylation sites of Tir at Y454 and Y474 are not required for blocking H. pylori-induced actin-cytoskeletal rearrangements. AGS cells were infected with H. pylori in the presence or absence of wt and isogenic EPEC mutant strains expressing the indicated Tir variants in EPECDtir. A. Inhibition of the H. pylori-induced host cell elongation by EPEC expressing either HA-tagged wt Tir or Tir mutated in the known phosphorylation sites, Y454 and/or Y474 respectively. The number of elongated AGS cells in each experiment was quantified in 10 different 0.25 mm2 fields. The capacity to induce elongation of infected AGS cells is represented as efficient induction (60–75% or + phenotype), drastically reduced or no induction (< 10%, or – phenotype). Infections were carried out for 4 h using different moi as indicated. Translocation-associated shifts in Tir’s apparent molecular mass was analysed by Western blotting using an a-HA antibody. Arrows indicate the different Tir forms on the gel. The size shift of Tir (asterisk) was reported to be dependent on PKA-mediated serine phosphorylation of Tir rather than its tyrosine phosphorylation (Warawa and Kenny, 2001). B. Tyrosine phosphorylation of CagA during co-infection was analysed by Western blotting using an a-phosphotyrosine antibody and an a-CagA antibody. C. Both EHEC-Tir and EPEC-Tir can block AGS cell elongation. As control, tyrosine phosphorylation of CagA during co-infection was also analysed by Western blotting using a-phosphotyrosine and a-CagA antibodies. Phosphotyrosine staining also indicates that EPEC Tir but not EHEC-Tir is tyrosine-phosphorylated. Arrows indicate CagA and HA-tagged Tir proteins on the gel. The results are representative from at least three independent experiments. D. Scanning electron microscopy of AGS cells infected with EPECDtir expressing the indicated Tir variants. This includes wt Tir from EPEC as well as EPEC-Tir mutated in the known phosphorylation sites, Y474 and/or Y454 respectively. Pedestal formation was only seen in EPEC-Tir that contain a functional Y474 (arrows). E. Merged immunofluorescence micrographs of infected AGS cells. EPEC was visualized by nuclear DNA-staining (DAPI; blue), filamentous actin (rhodamine-phalloidin; red) and tyrosine-phosphorylated proteins with an a-phosphotyrosine antibody (green, arrowheads). Arrows indicate typical actin pedestals. Bars in D and E, 2 mm.

Article Snippet: Cell culture, transient transfection and infection assays AGS cells (ATCC CRL 1739a human gastric adenocarcinoma epithelial cell line) were grown in 6 well plates containing RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) supplemented with 25 mM Hepes buffer and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany) for 2 days to reach monolayers of approximately 70% cell confluence.

Techniques: Phospho-proteomics, Blocking Assay, Infection, Mutagenesis, Expressing, Inhibition, Translocation Assay, Western Blot, Control, Staining, Electron Microscopy, Functional Assay

Journal: Cellular microbiology

Article Title: Dual infection system identifies a crucial role for PKA-mediated serine phosphorylation of the EPEC-Tir-injected effector protein in regulating Rac1 function.

doi: 10.1111/j.1462-5822.2009.01330.x

Figure Lengend Snippet: Fig. 5. The serine phosphorylation sites of Tir at S434 and S463 are required for blocking H. pylori-induced actin-cytoskeletal rearrangements. A. Schematic representation of Tir constructs used in this experiment. Numbers indicate the amino acid residue positions corresponding to the serine residue substitutions made in Tir. The two transmembrane domains (TM) flanking the Intimin-binding domain (IBD) and the two known tyrosine (Y454 and Y474) and serine (S434 and S463) phosphorylation sites are indicated. B. AGS cells were infected with H. pylori in the presence or absence of wt and isogenic EPECDtir mutant strains expressing the indicated Tir variants. The number of elongated AGS cells in each co-infection experiment was quantified in 10 different 0.25 mm2 fields. Infections were carried out for 4 h using moi = 100 (H. pylori) or moi = 40 (EPEC). The results are representative from at least three independent experiments. P-values of <0.005 (**) were considered as significant. C. Scanning electron microscopy of AGS cells infected with EPECDtir expressing the indicated Tir variants. The mutant bacteria bind very tightly to the cells similar to EPEC expressing wt Tir as shown in Fig. 4D. However, pedestal elongation was largely blocked by the serine-phosphorylation mutants. Bars, 2 mm.

Article Snippet: Cell culture, transient transfection and infection assays AGS cells (ATCC CRL 1739a human gastric adenocarcinoma epithelial cell line) were grown in 6 well plates containing RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) supplemented with 25 mM Hepes buffer and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany) for 2 days to reach monolayers of approximately 70% cell confluence.

Techniques: Phospho-proteomics, Blocking Assay, Construct, Residue, Binding Assay, Infection, Mutagenesis, Expressing, Electron Microscopy, Bacteria

Journal: Cellular microbiology

Article Title: Dual infection system identifies a crucial role for PKA-mediated serine phosphorylation of the EPEC-Tir-injected effector protein in regulating Rac1 function.

doi: 10.1111/j.1462-5822.2009.01330.x

Figure Lengend Snippet: Fig. 6. The serine phosphorylation sites of Tir at S434 and S463 are required for maximal activation of PKA. A. AGS cells were infected with H. pylori in the presence or absence of wt and isogenic EPECDtir mutant strains expressing the indicated Tir variants. Infections were carried out for 4 h using moi = 100 (H. pylori) or moi = 40 (EPEC). Similar levels of Tir were confirmed by Western blotting using an a-Tir antibody. Arrows indicate the different Tir variants on the gel. The size shift of Tir (asterisk) was reported to be dependent on PKA-mediated serine phosphorylation of Tir rather than its tyrosine phosphorylation (Warawa and Kenny, 2001). B. As control, tyrosine phosphorylation of CagA during co-infection was also analysed by Western blotting using a-phosphotyrosine and a-CagA antibodies respectively. C. Infection of AGS cells with EPEC induces the phosphorylation of PKA at T197 as detected by using a phospho-specific antibody. PKA activation was largely attenuated in infections with EPEC expressing Tir with S434/463A mutations and in the presence of the PKA-inhibitor PKI. The GAPDH blot served as loading control. The results are representative from at least three independent experiments. Quantification of PKA kinase activity (bottom panel) was performed by densitometric evaluation of the bands using the luminescence image analyser. The sample having the strongest phospho-signal corresponds to 100% kinase phosphorylation/ activity in the respective blot. P-values of < 0.005 (**) and < 0.05 (*) were considered as significant.

Article Snippet: Cell culture, transient transfection and infection assays AGS cells (ATCC CRL 1739a human gastric adenocarcinoma epithelial cell line) were grown in 6 well plates containing RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) supplemented with 25 mM Hepes buffer and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany) for 2 days to reach monolayers of approximately 70% cell confluence.

Techniques: Phospho-proteomics, Activation Assay, Infection, Mutagenesis, Expressing, Western Blot, Control, Activity Assay

Journal: Cellular microbiology

Article Title: Dual infection system identifies a crucial role for PKA-mediated serine phosphorylation of the EPEC-Tir-injected effector protein in regulating Rac1 function.

doi: 10.1111/j.1462-5822.2009.01330.x

Figure Lengend Snippet: Fig. 8. EPEC induces PKA-mediated inactivation of Rac1 by phosphorylating its serine residue 71 (S71). A. Infection of AGS cells with EPEC induces the phosphorylation of Rac1 at S71 as detected by a phospho-specific antibody (NEB Cell Signalling). While H. pylori-infected cells did not show phosphorylation of Rac1 at S71, EPEC induced high levels of phosphorylated Rac1. Phosphorylation of Rac1 was largely attenuated in infections with EPEC expressing Tir with S434/463A mutations and in the presence of the PKA-inhibitor PKI suggesting that activated PKA is directly involved in inactivating Rac1 by phosphorylating S71. B. Quantification of Rac1 phosphorylation was performed by densitometric evaluation of the bands using the luminescence image analyser. The sample having the strongest signal corresponds to 100% phosphorylation in the respective blot. The results are representative from at least three independent experiments. P-values of <0.005 (**) or <0.05 (*) were considered as significant.

Article Snippet: Cell culture, transient transfection and infection assays AGS cells (ATCC CRL 1739a human gastric adenocarcinoma epithelial cell line) were grown in 6 well plates containing RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) supplemented with 25 mM Hepes buffer and 10% heat-inactivated fetal bovine serum (FBS, Biochrom, Berlin, Germany) for 2 days to reach monolayers of approximately 70% cell confluence.

Techniques: Residue, Infection, Phospho-proteomics, Expressing

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