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    ATCC hep 2 cells atcc ccl 23
    Cellular distribution and orientation of GFP-RSV F tail in nonpolarized epithelial cells. (a) <t>HEp-2</t> cells were infected with RSV for 48 h to determine the cellular distribution of the F protein. RSV F (red) was distributed in a perinuclear manner, consistent
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    Cellular distribution and orientation of GFP-RSV F tail in nonpolarized epithelial cells. (a) HEp-2 cells were infected with RSV for 48 h to determine the cellular distribution of the F protein. RSV F (red) was distributed in a perinuclear manner, consistent

    Journal:

    Article Title: The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence

    doi: 10.1128/JVI.79.19.12528-12535.2005

    Figure Lengend Snippet: Cellular distribution and orientation of GFP-RSV F tail in nonpolarized epithelial cells. (a) HEp-2 cells were infected with RSV for 48 h to determine the cellular distribution of the F protein. RSV F (red) was distributed in a perinuclear manner, consistent

    Article Snippet: HEp-2 cells (ATCC CCL-23) were grown in Opti-MEM I medium (Invitrogen) supplemented with 2% fetal bovine serum, 320 μg/ml l -glutamine, 2.7 μg/ml amphotericin B, and 45 μg/ml gentamicin.

    Techniques: Infection

    ER and Golgi colocalization of GFP-RSV F tail constructs in nonpolarized HEp-2 cells. HEp-2 cells were transfected with GFP-RSV F tail (a and b) or GFP-RSV F tail (ΔCT) (c and d) to assess the codistribution of each protein with resident ER (a and

    Journal:

    Article Title: The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence

    doi: 10.1128/JVI.79.19.12528-12535.2005

    Figure Lengend Snippet: ER and Golgi colocalization of GFP-RSV F tail constructs in nonpolarized HEp-2 cells. HEp-2 cells were transfected with GFP-RSV F tail (a and b) or GFP-RSV F tail (ΔCT) (c and d) to assess the codistribution of each protein with resident ER (a and

    Article Snippet: HEp-2 cells (ATCC CCL-23) were grown in Opti-MEM I medium (Invitrogen) supplemented with 2% fetal bovine serum, 320 μg/ml l -glutamine, 2.7 μg/ml amphotericin B, and 45 μg/ml gentamicin.

    Techniques: Construct, Transfection

    Cellular distribution of RSV F opt in nonpolarized epithelial cells. (a) Schematic representation of full-length and mutant forms of RSV F opt . (b to e) HEp-2 cells were transfected with each RSV F opt construct noted in panel a to assess the cellular distribution

    Journal:

    Article Title: The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence

    doi: 10.1128/JVI.79.19.12528-12535.2005

    Figure Lengend Snippet: Cellular distribution of RSV F opt in nonpolarized epithelial cells. (a) Schematic representation of full-length and mutant forms of RSV F opt . (b to e) HEp-2 cells were transfected with each RSV F opt construct noted in panel a to assess the cellular distribution

    Article Snippet: HEp-2 cells (ATCC CCL-23) were grown in Opti-MEM I medium (Invitrogen) supplemented with 2% fetal bovine serum, 320 μg/ml l -glutamine, 2.7 μg/ml amphotericin B, and 45 μg/ml gentamicin.

    Techniques: Mutagenesis, Transfection, Construct

    Localization of the N-terminal deletion variants of CNF Y in the late endosome. HEp-2 cells were intoxicated with 500 nM CNF Y 1-1014-GFP, CNF Y Δ39-134-GFP or CNF Y Δ39-426-GFP green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and insets are magnified views of boxed areas. White scale bar is 10 µm.

    Journal: bioRxiv

    Article Title: The structure of bacterial toxin CNFY reveals requirements for secretion, host cell recognition and endosomal release

    doi: 10.1101/2020.04.07.029181

    Figure Lengend Snippet: Localization of the N-terminal deletion variants of CNF Y in the late endosome. HEp-2 cells were intoxicated with 500 nM CNF Y 1-1014-GFP, CNF Y Δ39-134-GFP or CNF Y Δ39-426-GFP green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents late endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and insets are magnified views of boxed areas. White scale bar is 10 µm.

    Article Snippet: HEp-2 cells (ATCC CCL-23) were grown at 37°C, 5% CO2 in RPMI (Gibco) supplemented with 7.5% newborn calf serum (NCS; Sigma).

    Techniques: Fluorescence, Microscopy, Staining

    Synthesis, secretion, host cell binding and effects of N-terminal deletion variants of CNF Y . A Schematic overview of the N-terminal CNF Y deletion variants. B C-terminally 3xFlag-tagged CNF Y and different N-terminally deleted toxin variants were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ) from plasmids under the control of their own promoter and were detected in whole cell extracts using an anti-Flag antibody. C To test secretion of the CNF Y variants, CNF Y and different N-terminally deleted variants fused to β-lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ). Beta-lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate. D Upper panel: HEp-2 cells remained untreated or were incubated with full-length CNF Y or the N-terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels; lower panel: HEp-2 cells were lysed and the cell extracts were incubated with full-length CNF Y or the N-terminally deleted toxin variants for 4 h. The deamidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE. E 3xFlag-tagged CNF Y and N-terminally deleted toxin variants were added to HEp-2 cells for 4 h. The cells were intensively washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody. F HEp-2 cells were incubated with 500 nM full-length CNF Y or the N-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNF Y -treated cells. The white scale bar is 40 µm.

    Journal: bioRxiv

    Article Title: The structure of bacterial toxin CNFY reveals requirements for secretion, host cell recognition and endosomal release

    doi: 10.1101/2020.04.07.029181

    Figure Lengend Snippet: Synthesis, secretion, host cell binding and effects of N-terminal deletion variants of CNF Y . A Schematic overview of the N-terminal CNF Y deletion variants. B C-terminally 3xFlag-tagged CNF Y and different N-terminally deleted toxin variants were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ) from plasmids under the control of their own promoter and were detected in whole cell extracts using an anti-Flag antibody. C To test secretion of the CNF Y variants, CNF Y and different N-terminally deleted variants fused to β-lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ). Beta-lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate. D Upper panel: HEp-2 cells remained untreated or were incubated with full-length CNF Y or the N-terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified Rho GTPase band in SDS PAGE gels; lower panel: HEp-2 cells were lysed and the cell extracts were incubated with full-length CNF Y or the N-terminally deleted toxin variants for 4 h. The deamidation of RhoA in the cell extracts was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE. E 3xFlag-tagged CNF Y and N-terminally deleted toxin variants were added to HEp-2 cells for 4 h. The cells were intensively washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody. F HEp-2 cells were incubated with 500 nM full-length CNF Y or the N-terminally deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of thick actin stress fibers and membrane actin folding were only observed with CNF Y -treated cells. The white scale bar is 40 µm.

    Article Snippet: HEp-2 cells (ATCC CCL-23) were grown at 37°C, 5% CO2 in RPMI (Gibco) supplemented with 7.5% newborn calf serum (NCS; Sigma).

    Techniques: Binding Assay, Transmission Electron Microscopy, Activity Assay, Incubation, Modification, SDS Page, Mobility Shift, Western Blot, Staining, Fluorescence, Microscopy

    Synthesis, secretion, host cell binding and effects of C-terminal deletion variants of CNF Y . A C-terminally 3xFlag-tagged CNF Y and different C-terminally deleted toxin variants were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ) from plasmids under control of their own promoter and were detected in whole cell extracts using an anti-Flag antibody. B HEp-2 cells remained untreated or were incubated with full-length CNF Y or the C-terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE. C HEp-2 cells were incubated with 500 nM full-length CNF Y or the C-terminal deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of actin stress fibers and membrane actin folding were only observed with full-length-CNF Y -treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (Δ cnfY ) harboring the empty expression vector were used as negative controls. D HEp-2 cells were incubated with 500 nM full-length CNF Y or the C-terminally deleted toxin variants fused to beta-lactamase (TEM) for 4 h. Cleavage of the reporter dye CCF4-AM was used to visualize toxin delivery. After cell entry CCF4-AM is rapidly converted into the negatively charged form CCF4, which is retained in the cytosol and emits a green fluorescence signal (520 nm). In the presence of translocated beta-lactamase fusion proteins, CCF4-AM is cleaved, and disruption of FRET results in blue fluorescence (447 nm). White bar: 20 µm. E 3xFlag-tagged CNF1, CNF Y and C-terminally deleted toxin variants were added to HEp-2 cells for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody. F To test secretion of the CNF Y variants, CNF Y and different C-terminally deleted variants fused to beta-lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ). Beta-lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate.

    Journal: bioRxiv

    Article Title: The structure of bacterial toxin CNFY reveals requirements for secretion, host cell recognition and endosomal release

    doi: 10.1101/2020.04.07.029181

    Figure Lengend Snippet: Synthesis, secretion, host cell binding and effects of C-terminal deletion variants of CNF Y . A C-terminally 3xFlag-tagged CNF Y and different C-terminally deleted toxin variants were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ) from plasmids under control of their own promoter and were detected in whole cell extracts using an anti-Flag antibody. B HEp-2 cells remained untreated or were incubated with full-length CNF Y or the C-terminally deleted toxin variants for 4 h. Cells were lysed and the deamidation of RhoA was analyzed by the mobility shift of the modified Rho GTPase on SDS PAGE. C HEp-2 cells were incubated with 500 nM full-length CNF Y or the C-terminal deleted toxin variants for 24 h. The cell nuclei were strained with DAPI (blue) and the actin cytoskeleton was stained using FITC-phalloidin (green). The formation of large, multinuclear cells was observed by fluorescence microscopy and the formation of actin stress fibers and membrane actin folding were only observed with full-length-CNF Y -treated cells. The white scale bar is 40 µm. Cells incubated with extracts of YP147 (Δ cnfY ) harboring the empty expression vector were used as negative controls. D HEp-2 cells were incubated with 500 nM full-length CNF Y or the C-terminally deleted toxin variants fused to beta-lactamase (TEM) for 4 h. Cleavage of the reporter dye CCF4-AM was used to visualize toxin delivery. After cell entry CCF4-AM is rapidly converted into the negatively charged form CCF4, which is retained in the cytosol and emits a green fluorescence signal (520 nm). In the presence of translocated beta-lactamase fusion proteins, CCF4-AM is cleaved, and disruption of FRET results in blue fluorescence (447 nm). White bar: 20 µm. E 3xFlag-tagged CNF1, CNF Y and C-terminally deleted toxin variants were added to HEp-2 cells for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody. F To test secretion of the CNF Y variants, CNF Y and different C-terminally deleted variants fused to beta-lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (Δ cnfY ). Beta-lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate.

    Article Snippet: HEp-2 cells (ATCC CCL-23) were grown at 37°C, 5% CO2 in RPMI (Gibco) supplemented with 7.5% newborn calf serum (NCS; Sigma).

    Techniques: Binding Assay, Incubation, Mobility Shift, Modification, SDS Page, Staining, Fluorescence, Microscopy, Expressing, Plasmid Preparation, Transmission Electron Microscopy, Western Blot, Activity Assay

    Localization of the C-terminal deletion variants of CNF Y in the late endosome. HEp-2 cells were intoxicated with 500 nM CNF Y 1-1014-GFP, CNF Y 1-719-GFP, CNF Y 1-526-GFP or CNF Y 1-443-GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents early endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and insets are magnified views of boxed areas. White scale bar is 10 µm.

    Journal: bioRxiv

    Article Title: The structure of bacterial toxin CNFY reveals requirements for secretion, host cell recognition and endosomal release

    doi: 10.1101/2020.04.07.029181

    Figure Lengend Snippet: Localization of the C-terminal deletion variants of CNF Y in the late endosome. HEp-2 cells were intoxicated with 500 nM CNF Y 1-1014-GFP, CNF Y 1-719-GFP, CNF Y 1-526-GFP or CNF Y 1-443-GFP (green) for 90 or 180 min. Cells were fixed and processed for fluorescence microscopy. The red fluorescent signal represents early endosomes (CellLight Late Endosomes-RFP (Rab7a)). Nuclei were stained with DAPI (blue). A merged image of the different channels is shown, and insets are magnified views of boxed areas. White scale bar is 10 µm.

    Article Snippet: HEp-2 cells (ATCC CCL-23) were grown at 37°C, 5% CO2 in RPMI (Gibco) supplemented with 7.5% newborn calf serum (NCS; Sigma).

    Techniques: Fluorescence, Microscopy, Staining

    2-D gel analysis. (A) HEp-2 cell 2-D profile of rifaximin (RX)-treated vs. untreated cells. Spots decreased in RX vs. untreated cells are outlined in blue and up-regulated spots in RX vs. untreated are outlined in red. See Table 1 for spot measurements. (B) HEp-2 cell 2-D profile of RX-treated vs. rifamycin (MY)-treated cells. Spots decreased in RX vs. MY-treated cells are outlined in blue and up-regulated spots in RX-treated vs. MY-treated are outlined in red. See Table 1 for spot measurements.

    Journal: PLoS ONE

    Article Title: Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis

    doi: 10.1371/journal.pone.0068550

    Figure Lengend Snippet: 2-D gel analysis. (A) HEp-2 cell 2-D profile of rifaximin (RX)-treated vs. untreated cells. Spots decreased in RX vs. untreated cells are outlined in blue and up-regulated spots in RX vs. untreated are outlined in red. See Table 1 for spot measurements. (B) HEp-2 cell 2-D profile of RX-treated vs. rifamycin (MY)-treated cells. Spots decreased in RX vs. MY-treated cells are outlined in blue and up-regulated spots in RX-treated vs. MY-treated are outlined in red. See Table 1 for spot measurements.

    Article Snippet: Cell Culture HEp-2 cells (larynx-derived cell line obtained from the American Type Culture collection ) were cultured in DMEM 10% fetal bovine serum media and grown to confluency as described then pretreated with rifaximin (RX), acetone (diluent control), or rifamycin (MY) (antibiotic control), or left untreated for 48 h. The media was removed, the cells washed 3X with phosphate buffered saline (PBS, pH 7.4), and the cells removed using a cell scraper.

    Techniques:

    FDG inhibits Stx transport to the ER and the release of StxA 1 A and B. HEp-2-GalT-GFP-SNAP cells were treated with 1 mM FDG for 4 h, or with 2 μg/ml BFA for 30 min prior to incubation with 125 I-Stx1-mut-BG for 1 h in the presence of the drugs. Cells were lysed, and Stx-SNAP was immunoprecipitated and run on SDS-PAGE. (A) The quantification of StxBbound to SNAP-tag. A representative autoradiogram is shown in (B). C. HEp-2-GalT-GFP-SNAP cells were treated with FDG as in (A) and (B) prior to incubation with Stx1-mut-BG-Alexa555. The cells were fixed, permeabilized and immunolabeled for giantin, prior to mounting with Prolong®Gold with DAPI, and imaged. The overlay image shows GalT-GFP-SNAP signal in green, giantin in red, Stx in grey and DAPI in blue. Scale bar, 10 μm. D and E. HEp2-ER-GFP-SNAP cells were treated as in (A) and (B) prior to incubation with 125 I-Stx1-mut-BG for 5 h. Cells were lysed and Stx-SNAP was immunoprecipitated and run on SDS-PAGE. (D) The quantification of StxB bound to SNAP-tag. A representative autoradiogram is shown in (E). F. HEp-2-ER-GFP-SNAP cells were treated with FDG as in (A) and (B), and incubated with Stx1-mut-BG-Alexa555 for 1 h, followed by 4 h incubation with fresh medium. Cells were fixed, permeabilized and immunolabeled for PDI, prior to mounting with Prolong®Gold with DAPI, and imaged. The overlay image shows ER-GFP-SNAP signal in green, PDI in red, Stx in grey and DAPI in blue. Scale bar, 10 μm. G and H. HEp-2 cells were treated with 1 mM FDG for 4 h prior to incubation with 125 I-Stx1-mut for 5 h at 37°C. Cell lysates were separated by reducing (+DTT) or non-reducing (−DTT) SDS-PAGE to determine the nicking of StxA or StxA 1 release, respectively. A representative autoradiogram is shown in (H). The amount of released or nicked StxA 1 was calculated as a percentage of total Stx (the sum of StxA and StxA 1 ). (A), (D) and (G) show mean +SEM from at least three independent experiments; *p

    Journal: Oncotarget

    Article Title: Cellular effects of fluorodeoxyglucose: Global changes in the lipidome and alteration in intracellular transport

    doi: 10.18632/oncotarget.13089

    Figure Lengend Snippet: FDG inhibits Stx transport to the ER and the release of StxA 1 A and B. HEp-2-GalT-GFP-SNAP cells were treated with 1 mM FDG for 4 h, or with 2 μg/ml BFA for 30 min prior to incubation with 125 I-Stx1-mut-BG for 1 h in the presence of the drugs. Cells were lysed, and Stx-SNAP was immunoprecipitated and run on SDS-PAGE. (A) The quantification of StxBbound to SNAP-tag. A representative autoradiogram is shown in (B). C. HEp-2-GalT-GFP-SNAP cells were treated with FDG as in (A) and (B) prior to incubation with Stx1-mut-BG-Alexa555. The cells were fixed, permeabilized and immunolabeled for giantin, prior to mounting with Prolong®Gold with DAPI, and imaged. The overlay image shows GalT-GFP-SNAP signal in green, giantin in red, Stx in grey and DAPI in blue. Scale bar, 10 μm. D and E. HEp2-ER-GFP-SNAP cells were treated as in (A) and (B) prior to incubation with 125 I-Stx1-mut-BG for 5 h. Cells were lysed and Stx-SNAP was immunoprecipitated and run on SDS-PAGE. (D) The quantification of StxB bound to SNAP-tag. A representative autoradiogram is shown in (E). F. HEp-2-ER-GFP-SNAP cells were treated with FDG as in (A) and (B), and incubated with Stx1-mut-BG-Alexa555 for 1 h, followed by 4 h incubation with fresh medium. Cells were fixed, permeabilized and immunolabeled for PDI, prior to mounting with Prolong®Gold with DAPI, and imaged. The overlay image shows ER-GFP-SNAP signal in green, PDI in red, Stx in grey and DAPI in blue. Scale bar, 10 μm. G and H. HEp-2 cells were treated with 1 mM FDG for 4 h prior to incubation with 125 I-Stx1-mut for 5 h at 37°C. Cell lysates were separated by reducing (+DTT) or non-reducing (−DTT) SDS-PAGE to determine the nicking of StxA or StxA 1 release, respectively. A representative autoradiogram is shown in (H). The amount of released or nicked StxA 1 was calculated as a percentage of total Stx (the sum of StxA and StxA 1 ). (A), (D) and (G) show mean +SEM from at least three independent experiments; *p

    Article Snippet: Cells HEp-2 cells (ATCC/LGC, CCL-23) were cultured in DMEM+GlutaMAX™ (Gibco) medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Incubation, Immunoprecipitation, SDS Page, Immunolabeling

    LPL treatment effects on Stx cell association. HEp-2 cells were pretreated with LPLs or other substances for 30 min in HEPES-buffered medium without FBS for 30 min at 37 °C, prior to incubation with 125 I-Stx1m for 20 min at 37 °C. Cells were washed, lysed and the radioactivity was measured. All panels show quantification data of cell-associated 125 I-Stx1m, expressed as % of control. (a) Cells were pretreated with 10 or 20 μM LPLs before incubation with 125 I-Stx1m (mean ± SEM; n ≥ 3, except for 20 μM LPS brain and 20 μM LPE brain plasmalogen showing mean ± STD; n = 2). (b) A representative figure for one out of five independent experiments, showing amounts of cell-associated 125 I-Stx1m when treated with LPI (bovine liver LPI, mostly 18:0), pure LPI 18:0 and pure LPI 16:0 at increasing concentrations (mean ± STD, duplicates). (c) Quantification of cell-associated 125 I-Stx1m after treatment with LPI (bovine liver, mostly 18:0), LPI 18:0 and LPI 16:0 at increasing concentrations (mean ± SEM; n ≥ 5). (d) Quantification of cell-associated 125 I-Stx1m after treatment with LPC 18:0 and LPC 18:1 at 10 and 20 μM (mean ± SEM; n = 3). (e) Quantification of cell-associated 125 I-Stx1m upon treatment with various LPLs with acyl chains containing mostly 18:0 or pure 18:0 (mean ± SEM; n ≥ 3).

    Journal: Scientific Reports

    Article Title: Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding

    doi: 10.1038/srep30336

    Figure Lengend Snippet: LPL treatment effects on Stx cell association. HEp-2 cells were pretreated with LPLs or other substances for 30 min in HEPES-buffered medium without FBS for 30 min at 37 °C, prior to incubation with 125 I-Stx1m for 20 min at 37 °C. Cells were washed, lysed and the radioactivity was measured. All panels show quantification data of cell-associated 125 I-Stx1m, expressed as % of control. (a) Cells were pretreated with 10 or 20 μM LPLs before incubation with 125 I-Stx1m (mean ± SEM; n ≥ 3, except for 20 μM LPS brain and 20 μM LPE brain plasmalogen showing mean ± STD; n = 2). (b) A representative figure for one out of five independent experiments, showing amounts of cell-associated 125 I-Stx1m when treated with LPI (bovine liver LPI, mostly 18:0), pure LPI 18:0 and pure LPI 16:0 at increasing concentrations (mean ± STD, duplicates). (c) Quantification of cell-associated 125 I-Stx1m after treatment with LPI (bovine liver, mostly 18:0), LPI 18:0 and LPI 16:0 at increasing concentrations (mean ± SEM; n ≥ 5). (d) Quantification of cell-associated 125 I-Stx1m after treatment with LPC 18:0 and LPC 18:1 at 10 and 20 μM (mean ± SEM; n = 3). (e) Quantification of cell-associated 125 I-Stx1m upon treatment with various LPLs with acyl chains containing mostly 18:0 or pure 18:0 (mean ± SEM; n ≥ 3).

    Article Snippet: Cell lines HEp-2 cells (ATCC: CCL-23, 1994) and HeLa (Institute Curie, Paris, France, 2002) were grown in a humidified 5% CO2 atmosphere at 37 °C and maintained in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; D0819, Sigma-Aldrich) supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin.

    Techniques: Incubation, Radioactivity

    LPL treatment changes plasma membrane lipid packing. (a) HEp-2 cells were treated with 10 μM LPI, LPE, LPC 18:0, LPC 18:1 or 0.2% ethanol (EtOH) for 20 min, or with 5 mM mβCD for 45 min, prior to staining with 10 nM NR12S. Spectral imaging was started 7 min after the addition of NR12S and continued for up to 15 min. The analysis of the spectra was performed on at least 20 manually selected regions of the plasma membrane for each condition. The panel to the left shows the data from one of three independent experiments with LPI, LPE and mβCD and the panel to the right shows data from one of two independent experiments with LPC 18:0 and LPC 18:1 (mean ± STD). (b) The average GP value for each selection was quantified as described in materials and methods, and the figure shows differences in GP values between the EtOH and the treated samples (mean ± SEM; n ≥ 2). (c) Pseudo-coloured TIRF images of the basal plasma membrane following cell treatment with 0.2% EtOH or 10 μM LPI. The dark blue structures are the coating of the Lab-Tek dish stained by NR12S. The colour scale for GP values is shown on the right; scale bar 10 μm. (d) Differential interference contrast images showing morphology of HEp-2 cells, taken 10 min prior to and 10 min after addition of 0.2% EtOH, 10 μM LPI or 10 μM LPE; scale bar 10 μm.

    Journal: Scientific Reports

    Article Title: Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding

    doi: 10.1038/srep30336

    Figure Lengend Snippet: LPL treatment changes plasma membrane lipid packing. (a) HEp-2 cells were treated with 10 μM LPI, LPE, LPC 18:0, LPC 18:1 or 0.2% ethanol (EtOH) for 20 min, or with 5 mM mβCD for 45 min, prior to staining with 10 nM NR12S. Spectral imaging was started 7 min after the addition of NR12S and continued for up to 15 min. The analysis of the spectra was performed on at least 20 manually selected regions of the plasma membrane for each condition. The panel to the left shows the data from one of three independent experiments with LPI, LPE and mβCD and the panel to the right shows data from one of two independent experiments with LPC 18:0 and LPC 18:1 (mean ± STD). (b) The average GP value for each selection was quantified as described in materials and methods, and the figure shows differences in GP values between the EtOH and the treated samples (mean ± SEM; n ≥ 2). (c) Pseudo-coloured TIRF images of the basal plasma membrane following cell treatment with 0.2% EtOH or 10 μM LPI. The dark blue structures are the coating of the Lab-Tek dish stained by NR12S. The colour scale for GP values is shown on the right; scale bar 10 μm. (d) Differential interference contrast images showing morphology of HEp-2 cells, taken 10 min prior to and 10 min after addition of 0.2% EtOH, 10 μM LPI or 10 μM LPE; scale bar 10 μm.

    Article Snippet: Cell lines HEp-2 cells (ATCC: CCL-23, 1994) and HeLa (Institute Curie, Paris, France, 2002) were grown in a humidified 5% CO2 atmosphere at 37 °C and maintained in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; D0819, Sigma-Aldrich) supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin.

    Techniques: Staining, Imaging, Selection

    LPI effects on Stx binding are independent of signalling and ATP. (a) HEp-2 cells were pretreated with LPI (10 μM) or GPR55 agonist O-1602 (10 μM) for 10 min prior to addition of Stx1m (100 ng/ml) for 30 min at 37 °C. Cells were washed, fixed, permeabilized and blocked in 5% FBS for 30 min prior to incubation with primary and secondary antibodies. Confocal microscopy images show Stx staining in white and nuclei in blue; scale bar 10 μm. (b) Cells were pretreated with or without 2-deoxy-D-glucose (50 mM) and NaN 3 (10 mM) for 1 h to deplete cellular ATP before 10 μM LPI was added and incubation was continued for 30 min. Cells were then cooled down for 10 min and 125 I-Stx1m was added for 20 min on ice. Cells were washed, lysed and the radioactivity signal was counted. Results are presented as % of control (mean ± STD; n = 3 for ctrl and n = 2 for LPI). (c) Cells were washed with HEPES-buffered medium to remove serum proteins and fixed with 10% formalin solution for 30 min. The fixed cells were treated with 10 μM LPI for 30 min at 37 °C and then incubated with 125 I-Stx1m for 20 min at 37 °C. Cell-associated 125 I-Stx1m was counted and presented as % of control (mean ± SEM; n = 3).

    Journal: Scientific Reports

    Article Title: Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding

    doi: 10.1038/srep30336

    Figure Lengend Snippet: LPI effects on Stx binding are independent of signalling and ATP. (a) HEp-2 cells were pretreated with LPI (10 μM) or GPR55 agonist O-1602 (10 μM) for 10 min prior to addition of Stx1m (100 ng/ml) for 30 min at 37 °C. Cells were washed, fixed, permeabilized and blocked in 5% FBS for 30 min prior to incubation with primary and secondary antibodies. Confocal microscopy images show Stx staining in white and nuclei in blue; scale bar 10 μm. (b) Cells were pretreated with or without 2-deoxy-D-glucose (50 mM) and NaN 3 (10 mM) for 1 h to deplete cellular ATP before 10 μM LPI was added and incubation was continued for 30 min. Cells were then cooled down for 10 min and 125 I-Stx1m was added for 20 min on ice. Cells were washed, lysed and the radioactivity signal was counted. Results are presented as % of control (mean ± STD; n = 3 for ctrl and n = 2 for LPI). (c) Cells were washed with HEPES-buffered medium to remove serum proteins and fixed with 10% formalin solution for 30 min. The fixed cells were treated with 10 μM LPI for 30 min at 37 °C and then incubated with 125 I-Stx1m for 20 min at 37 °C. Cell-associated 125 I-Stx1m was counted and presented as % of control (mean ± SEM; n = 3).

    Article Snippet: Cell lines HEp-2 cells (ATCC: CCL-23, 1994) and HeLa (Institute Curie, Paris, France, 2002) were grown in a humidified 5% CO2 atmosphere at 37 °C and maintained in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; D0819, Sigma-Aldrich) supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin.

    Techniques: Binding Assay, Incubation, Confocal Microscopy, Staining, Radioactivity

    LPL effects on toxin cell association, transport and toxicity. (a) HEp-2 cells were pretreated with 10 μM LPLs with acyl chains consisting of pure or mostly C18:0 for 30 min, prior to incubation with 125 I-Stx2 for 20 min at 37 °C. Cells were washed, lysed and the radioactivity was measured. Cell-associated 125 I-Stx2 is expressed as % of control (mean ± deviation; n = 2). (b) Sulfation assay to measure the amounts of StxBSulf2 transported to the Golgi apparatus where the modified toxin is sulfated. HEp-2 cells were pre-incubated with 35 SO 4 2− for 1.5 h and then incubated with 10 μM LPLs with acyl chains consisting of pure or mostly C18:0 for 30 min, followed by addition of 2 μg/ml StxBS2 for 1.5 h. Shiga toxin was immunoprecipitated from cell lysates and radioactivity was counted. Results are shown as % of ctrl (mean ± SEM; n = 3). N/A stands for not applicable (below detection limit). (c,d) Toxicity assay with Shiga holotoxin (Stx) and Stx2. Cells were pretreated with 5 μM LPLs with acyl chains consisting of pure or mostly C18:0 for 30 min, followed by incubation with tenfold serial toxin dilutions for 4 h in leucine-free medium. Protein synthesis was measured and results are shown as % of no toxin (mean ± deviation; n = 2 for each of the toxins).

    Journal: Scientific Reports

    Article Title: Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding

    doi: 10.1038/srep30336

    Figure Lengend Snippet: LPL effects on toxin cell association, transport and toxicity. (a) HEp-2 cells were pretreated with 10 μM LPLs with acyl chains consisting of pure or mostly C18:0 for 30 min, prior to incubation with 125 I-Stx2 for 20 min at 37 °C. Cells were washed, lysed and the radioactivity was measured. Cell-associated 125 I-Stx2 is expressed as % of control (mean ± deviation; n = 2). (b) Sulfation assay to measure the amounts of StxBSulf2 transported to the Golgi apparatus where the modified toxin is sulfated. HEp-2 cells were pre-incubated with 35 SO 4 2− for 1.5 h and then incubated with 10 μM LPLs with acyl chains consisting of pure or mostly C18:0 for 30 min, followed by addition of 2 μg/ml StxBS2 for 1.5 h. Shiga toxin was immunoprecipitated from cell lysates and radioactivity was counted. Results are shown as % of ctrl (mean ± SEM; n = 3). N/A stands for not applicable (below detection limit). (c,d) Toxicity assay with Shiga holotoxin (Stx) and Stx2. Cells were pretreated with 5 μM LPLs with acyl chains consisting of pure or mostly C18:0 for 30 min, followed by incubation with tenfold serial toxin dilutions for 4 h in leucine-free medium. Protein synthesis was measured and results are shown as % of no toxin (mean ± deviation; n = 2 for each of the toxins).

    Article Snippet: Cell lines HEp-2 cells (ATCC: CCL-23, 1994) and HeLa (Institute Curie, Paris, France, 2002) were grown in a humidified 5% CO2 atmosphere at 37 °C and maintained in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; D0819, Sigma-Aldrich) supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin.

    Techniques: Incubation, Radioactivity, Modification, Immunoprecipitation

    Characterization of LPI-induced inhibition of Stx binding to Gb3. (a) HEp-2 cells were pretreated with increasing concentrations of LPI for 30 min at 37 °C or on ice, prior to cooling down and incubation with 125 I-Stx1m for 30 min on ice. Cells were washed, lysed and the radioactive signal was measured. Results are presented as % of control (mean ± SEM; n = 3). (b) Cells were pretreated with increasing concentrations of LPI for 30 min at 37 °C, prior to incubation with 125 I-Stx1m for 20 min at 37 °C. Cells were washed, lysed and the radioactive signal was measured, results are presented as % of control (mean ± SEM; n = 3). (c) Cells were pretreated with 10 μM LPI for 30 min in absence or presence of 10% FBS prior to incubation with 125 I-Stx1m for 20 min at 37 °C. The quantification of radioactivity measurements is shown, presented as % of control (mean ± SEM; n = 3). (d) Cells were incubated with 10 μM LPI for 30 min and then washed once at 37 °C with HEPES-buffered cell growth medium with or without 10% FBS, prior to cooling down on ice for 10 min and incubation with 125 I-Stx1m on ice for 20 min. The quantification of radioactivity measurements is shown, presented as % of control (mean ± STD; n = 2). (e) Cells were pretreated with 10 μM LPI for 30 min prior to addition of Stx1m (100 ng/ml) or anti-Gb3 antibody and incubated for 20 min at 37 °C. Cells were washed, fixed, permeabilized and blocked in 5% FBS for 1 hr prior to incubation with primary (Stx samples only) and secondary antibodies. Confocal microscopy images show Stx or anti-Gb3 antibody staining in white and DAPI nuclear staining in blue; scale bar 10 μm. (f) Quantification of intensity signals of Stx and anti-Gb3 antibodies from confocal images, expressed as % of control (mean ± SEM; n = 3).

    Journal: Scientific Reports

    Article Title: Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding

    doi: 10.1038/srep30336

    Figure Lengend Snippet: Characterization of LPI-induced inhibition of Stx binding to Gb3. (a) HEp-2 cells were pretreated with increasing concentrations of LPI for 30 min at 37 °C or on ice, prior to cooling down and incubation with 125 I-Stx1m for 30 min on ice. Cells were washed, lysed and the radioactive signal was measured. Results are presented as % of control (mean ± SEM; n = 3). (b) Cells were pretreated with increasing concentrations of LPI for 30 min at 37 °C, prior to incubation with 125 I-Stx1m for 20 min at 37 °C. Cells were washed, lysed and the radioactive signal was measured, results are presented as % of control (mean ± SEM; n = 3). (c) Cells were pretreated with 10 μM LPI for 30 min in absence or presence of 10% FBS prior to incubation with 125 I-Stx1m for 20 min at 37 °C. The quantification of radioactivity measurements is shown, presented as % of control (mean ± SEM; n = 3). (d) Cells were incubated with 10 μM LPI for 30 min and then washed once at 37 °C with HEPES-buffered cell growth medium with or without 10% FBS, prior to cooling down on ice for 10 min and incubation with 125 I-Stx1m on ice for 20 min. The quantification of radioactivity measurements is shown, presented as % of control (mean ± STD; n = 2). (e) Cells were pretreated with 10 μM LPI for 30 min prior to addition of Stx1m (100 ng/ml) or anti-Gb3 antibody and incubated for 20 min at 37 °C. Cells were washed, fixed, permeabilized and blocked in 5% FBS for 1 hr prior to incubation with primary (Stx samples only) and secondary antibodies. Confocal microscopy images show Stx or anti-Gb3 antibody staining in white and DAPI nuclear staining in blue; scale bar 10 μm. (f) Quantification of intensity signals of Stx and anti-Gb3 antibodies from confocal images, expressed as % of control (mean ± SEM; n = 3).

    Article Snippet: Cell lines HEp-2 cells (ATCC: CCL-23, 1994) and HeLa (Institute Curie, Paris, France, 2002) were grown in a humidified 5% CO2 atmosphere at 37 °C and maintained in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; D0819, Sigma-Aldrich) supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin.

    Techniques: Inhibition, Binding Assay, Incubation, Radioactivity, Confocal Microscopy, Staining

    LPI releases prebound Stx; the inhibitory effect of LPI on Stx binding is reversed by mβCD; LPI has almost no inhibitory effect on binding of ricin. (a) HEp-2 cells were incubated with 125 I-Stx1m on ice for 30 min, washed and treated with 10 μM LPI for 20 min at 37 °C. Cell medium was collected, cells were lysed and the radioactivity signal was counted. The quantification of 125 I-Stx1m counts is expressed as % of total counts (mean ± SEM; n = 3). (b) Cells were pretreated with 10 μM LPI for 30 min and then increasing concentrations of mβCD were added and treatment continued for 15 min. Cells were then incubated with 125 I-Stx1m for 20 min at 37 °C and cell-associated 125 I-Stx1m was counted, quantification results presented as % of control (mean ± SEM; n = 3). (c) Cells were pretreated with 10 μM LPI for 30 min and then 10 mM mβCD was added and treatment continued for 15 min. Cells were then incubated with 125 I-Stx1m for 20 min on ice and cell bound 125 I-Stx1m was counted, quantification results presented as % of control (mean ± SEM; n = 3). (d) Cells were pretreated with 10 μM LPI for 30 min at 37 °C, prior to incubation with 125 I-ricin (20 min 37 °C). After the treatment, cells were washed, lysed and the radioactive signal was measured. Quantification data are presented as % of control (mean ± SEM; n = 6).

    Journal: Scientific Reports

    Article Title: Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding

    doi: 10.1038/srep30336

    Figure Lengend Snippet: LPI releases prebound Stx; the inhibitory effect of LPI on Stx binding is reversed by mβCD; LPI has almost no inhibitory effect on binding of ricin. (a) HEp-2 cells were incubated with 125 I-Stx1m on ice for 30 min, washed and treated with 10 μM LPI for 20 min at 37 °C. Cell medium was collected, cells were lysed and the radioactivity signal was counted. The quantification of 125 I-Stx1m counts is expressed as % of total counts (mean ± SEM; n = 3). (b) Cells were pretreated with 10 μM LPI for 30 min and then increasing concentrations of mβCD were added and treatment continued for 15 min. Cells were then incubated with 125 I-Stx1m for 20 min at 37 °C and cell-associated 125 I-Stx1m was counted, quantification results presented as % of control (mean ± SEM; n = 3). (c) Cells were pretreated with 10 μM LPI for 30 min and then 10 mM mβCD was added and treatment continued for 15 min. Cells were then incubated with 125 I-Stx1m for 20 min on ice and cell bound 125 I-Stx1m was counted, quantification results presented as % of control (mean ± SEM; n = 3). (d) Cells were pretreated with 10 μM LPI for 30 min at 37 °C, prior to incubation with 125 I-ricin (20 min 37 °C). After the treatment, cells were washed, lysed and the radioactive signal was measured. Quantification data are presented as % of control (mean ± SEM; n = 6).

    Article Snippet: Cell lines HEp-2 cells (ATCC: CCL-23, 1994) and HeLa (Institute Curie, Paris, France, 2002) were grown in a humidified 5% CO2 atmosphere at 37 °C and maintained in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; D0819, Sigma-Aldrich) supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin.

    Techniques: Binding Assay, Incubation, Radioactivity

    The decrease in c-Myc protein concentration can be ascribed to the interaction between IncA and G3BP1. (A) Schematic structure of the Cp. psittaci IncA (aa 121-383) double mutant (N159S; S237G) which is no longer able to interact with G3BP1 in the yeast two-hybrid system. Mutagenesis was performed in S. cerevisiae using error-prone PCR. (B) Yeast two-hybrid interaction assay of mutated IncA with G3BP1. Interactions were quantitatively assessed by β-galactosidase liquid culture activity assays. β-galactosidase values represent the mean numbers and standard deviations of three independent experiments. (1) GAL4 BD-IncA (aa 121-383)-EGFP + pGADT7 (negative control), (2) GAL4 BD-IncA (aa 121-383)-EGFP + GAL4 AD-G3BP1 (aa 221-451) (positive control), (3) GAL4 BD-IncA (aa 121-383, N159S; S237G)-EGFP + GAL4 AD-G3BP1 (aa 221-451). (C) Immunoblots of whole HEK293 cell lysates transfected with pcDNA3 (1), pcDNA3-HA-IncA (aa 121-383) (2) or pcDNA3-HA-IncA (aa 121-383, N159S; S237G) (3) and probed with anti-HA, anti-c-Myc and anti-β-actin antibodies. Bar diagram below the immunoblots represent a quantification of c-Myc protein concentration related to β-actin. Experiments were performed in triplicate. (D) Immunoblots of whole Hep-2 cells untreated (1), transfected with non-targeting siRNA (2) or transfected with ON-TARGET G3BP1 siRNA pool and probed with anti-G3BP1, anti-c-Myc and anti-β-actin antibodies. Bar diagrams show knock-down rate of G3BP1 and c-Myc standardized to β-actin and the c-myc mRNA concentrations estimated by qRT-PCR. Experiments were performed in triplicate.

    Journal: PLoS ONE

    Article Title: Functional Interaction between Type III-Secreted Protein IncA of Chlamydophila psittaci and Human G3BP1

    doi: 10.1371/journal.pone.0016692

    Figure Lengend Snippet: The decrease in c-Myc protein concentration can be ascribed to the interaction between IncA and G3BP1. (A) Schematic structure of the Cp. psittaci IncA (aa 121-383) double mutant (N159S; S237G) which is no longer able to interact with G3BP1 in the yeast two-hybrid system. Mutagenesis was performed in S. cerevisiae using error-prone PCR. (B) Yeast two-hybrid interaction assay of mutated IncA with G3BP1. Interactions were quantitatively assessed by β-galactosidase liquid culture activity assays. β-galactosidase values represent the mean numbers and standard deviations of three independent experiments. (1) GAL4 BD-IncA (aa 121-383)-EGFP + pGADT7 (negative control), (2) GAL4 BD-IncA (aa 121-383)-EGFP + GAL4 AD-G3BP1 (aa 221-451) (positive control), (3) GAL4 BD-IncA (aa 121-383, N159S; S237G)-EGFP + GAL4 AD-G3BP1 (aa 221-451). (C) Immunoblots of whole HEK293 cell lysates transfected with pcDNA3 (1), pcDNA3-HA-IncA (aa 121-383) (2) or pcDNA3-HA-IncA (aa 121-383, N159S; S237G) (3) and probed with anti-HA, anti-c-Myc and anti-β-actin antibodies. Bar diagram below the immunoblots represent a quantification of c-Myc protein concentration related to β-actin. Experiments were performed in triplicate. (D) Immunoblots of whole Hep-2 cells untreated (1), transfected with non-targeting siRNA (2) or transfected with ON-TARGET G3BP1 siRNA pool and probed with anti-G3BP1, anti-c-Myc and anti-β-actin antibodies. Bar diagrams show knock-down rate of G3BP1 and c-Myc standardized to β-actin and the c-myc mRNA concentrations estimated by qRT-PCR. Experiments were performed in triplicate.

    Article Snippet: Cell culture and organisms Hep-2 cells (ATCC-CCL23) were grown in DMEM medium with 4.5 g/l glucose (PAA) supplemented with 10% heat-inactivated fetal calf serum (FCS) and maintained at 37°C in an atmosphere of 5% CO2 .

    Techniques: Protein Concentration, Mutagenesis, Polymerase Chain Reaction, Activity Assay, Negative Control, Positive Control, Western Blot, Transfection, Quantitative RT-PCR

    G3BP1 is accumulated around the chlamydial inclusion in Cp. psittaci infected Hep-2 cells. Hep-2 cells were infected with Cp. psittaci at a MOI of 3, and at 48 h postinfection the cells were fixed and stained with anti-chlamydial LPS (red), anti-G3BP1 (green) antibodies and DAPI (blue, nuclei), and viewed under a fluorescence (1–3) or a confocal laser-scanning microscope (4–6). Non-infected cells served as control (1–3). The anti-G3BP1 (1 and 4) and anti-LPS (5) or DAPI (2) signals were merged (3 and 6). A fraction of endogenous G3BP1 is concentrated around chlamydial inclusions (6). Bar = 20 µm.

    Journal: PLoS ONE

    Article Title: Functional Interaction between Type III-Secreted Protein IncA of Chlamydophila psittaci and Human G3BP1

    doi: 10.1371/journal.pone.0016692

    Figure Lengend Snippet: G3BP1 is accumulated around the chlamydial inclusion in Cp. psittaci infected Hep-2 cells. Hep-2 cells were infected with Cp. psittaci at a MOI of 3, and at 48 h postinfection the cells were fixed and stained with anti-chlamydial LPS (red), anti-G3BP1 (green) antibodies and DAPI (blue, nuclei), and viewed under a fluorescence (1–3) or a confocal laser-scanning microscope (4–6). Non-infected cells served as control (1–3). The anti-G3BP1 (1 and 4) and anti-LPS (5) or DAPI (2) signals were merged (3 and 6). A fraction of endogenous G3BP1 is concentrated around chlamydial inclusions (6). Bar = 20 µm.

    Article Snippet: Cell culture and organisms Hep-2 cells (ATCC-CCL23) were grown in DMEM medium with 4.5 g/l glucose (PAA) supplemented with 10% heat-inactivated fetal calf serum (FCS) and maintained at 37°C in an atmosphere of 5% CO2 .

    Techniques: Infection, Staining, Fluorescence, Laser-Scanning Microscopy

    Infection of Hep-2 cells with Cp. psittaci leads to a decrease in c-Myc protein concentration. Hep-2 cells were infected with Cp. psittaci at a MOI of 3, with heat-inactivated Cp. psittaci or chloramphenicol-treated after infection as controls, and at different hours postinfection (hpi) cells were lysed for RNA or protein analysis. (A, above) Semi-quantitative (sq) PCR shows the unchanged expression of c-myc mRNA in infected (middle column), infected and chloramphenicol-treated (right) and control (left) Hep-2 cells. β-actin served as an internal control gene. (A, below) Quantitative Real-Time (qRT) PCR. c-myc levels were standardized on GAPDH . Experiments were performed three times in duplicate. (B) Immunoblots of whole cell lysates of Cp. psittaci infected (middle column), infected and chloramphenicol-treated (right) and control (left) cells probed with anti-LPS, anti-c-Myc and anti-β-actin antibodies. Bar diagrams below the immunoblots represent a quantification of c-Myc protein related to β-actin protein concentration. Experiments were performed three times.

    Journal: PLoS ONE

    Article Title: Functional Interaction between Type III-Secreted Protein IncA of Chlamydophila psittaci and Human G3BP1

    doi: 10.1371/journal.pone.0016692

    Figure Lengend Snippet: Infection of Hep-2 cells with Cp. psittaci leads to a decrease in c-Myc protein concentration. Hep-2 cells were infected with Cp. psittaci at a MOI of 3, with heat-inactivated Cp. psittaci or chloramphenicol-treated after infection as controls, and at different hours postinfection (hpi) cells were lysed for RNA or protein analysis. (A, above) Semi-quantitative (sq) PCR shows the unchanged expression of c-myc mRNA in infected (middle column), infected and chloramphenicol-treated (right) and control (left) Hep-2 cells. β-actin served as an internal control gene. (A, below) Quantitative Real-Time (qRT) PCR. c-myc levels were standardized on GAPDH . Experiments were performed three times in duplicate. (B) Immunoblots of whole cell lysates of Cp. psittaci infected (middle column), infected and chloramphenicol-treated (right) and control (left) cells probed with anti-LPS, anti-c-Myc and anti-β-actin antibodies. Bar diagrams below the immunoblots represent a quantification of c-Myc protein related to β-actin protein concentration. Experiments were performed three times.

    Article Snippet: Cell culture and organisms Hep-2 cells (ATCC-CCL23) were grown in DMEM medium with 4.5 g/l glucose (PAA) supplemented with 10% heat-inactivated fetal calf serum (FCS) and maintained at 37°C in an atmosphere of 5% CO2 .

    Techniques: Infection, Protein Concentration, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Western Blot

    Full-length IncA of Cp. psittaci and G3BP1 proteins interact in vitro and in vivo . (A) Purified GST, GST-IncA (aa 1-338) and GST-IncA (aa 121-383) or (C) GST and GST-G3BP1 (aa1-466) stained with Coomassie. (B) Results of GST pull-down experiments performed with IncA full-length and truncation clone fused to GST and [ 35 S]-methionine-labeled full-length G3BP1 or (E) with Hep-2 cell lysate instead of labeled G3BP1. (D) Results of GST pull-down experiment performed with full-length G3BP1 fused to GST and [ 35 S]-methionine-labeled IncA (aa 121-383). GST served as control. IVT: 10 µl of the in vitro translation product. Input: 25% (75 µg protein) of Hep-2 cell lysate used for the interaction assay. (F) Whole cell lysate of HEK293 cells transfected with a His-tagged codon-adapted IncA mammalian expression construct was subjected to co-immunoprecipitation experiments using anti-His antibody. Co-immunoprecipitated endogenous G3BP1 was detected with a mouse anti-G3BP1 monoclonal antibody. Controls were beads alone (c) or pre-immune serum (lgG). Input: 10% (100 µg) of the protein used for immunoprecipitation.

    Journal: PLoS ONE

    Article Title: Functional Interaction between Type III-Secreted Protein IncA of Chlamydophila psittaci and Human G3BP1

    doi: 10.1371/journal.pone.0016692

    Figure Lengend Snippet: Full-length IncA of Cp. psittaci and G3BP1 proteins interact in vitro and in vivo . (A) Purified GST, GST-IncA (aa 1-338) and GST-IncA (aa 121-383) or (C) GST and GST-G3BP1 (aa1-466) stained with Coomassie. (B) Results of GST pull-down experiments performed with IncA full-length and truncation clone fused to GST and [ 35 S]-methionine-labeled full-length G3BP1 or (E) with Hep-2 cell lysate instead of labeled G3BP1. (D) Results of GST pull-down experiment performed with full-length G3BP1 fused to GST and [ 35 S]-methionine-labeled IncA (aa 121-383). GST served as control. IVT: 10 µl of the in vitro translation product. Input: 25% (75 µg protein) of Hep-2 cell lysate used for the interaction assay. (F) Whole cell lysate of HEK293 cells transfected with a His-tagged codon-adapted IncA mammalian expression construct was subjected to co-immunoprecipitation experiments using anti-His antibody. Co-immunoprecipitated endogenous G3BP1 was detected with a mouse anti-G3BP1 monoclonal antibody. Controls were beads alone (c) or pre-immune serum (lgG). Input: 10% (100 µg) of the protein used for immunoprecipitation.

    Article Snippet: Cell culture and organisms Hep-2 cells (ATCC-CCL23) were grown in DMEM medium with 4.5 g/l glucose (PAA) supplemented with 10% heat-inactivated fetal calf serum (FCS) and maintained at 37°C in an atmosphere of 5% CO2 .

    Techniques: In Vitro, In Vivo, Purification, Staining, Labeling, Transfection, Expressing, Construct, Immunoprecipitation

    Hsp90 inhibitors have anti-RSV activity at non-toxic concentrations. (A–B) Toxicity of 17AAG (A) or 17DMAG (B) in HEp-2 cells was assessed after 72 hours using the Alamar blue cytotoxicity assay. (C–D) Hsp90 inhibitors reduce RSV replication. HEp-2 cells infected with RSV-A2 at an MOI of 0.01 were treated with the indicated concentrations of 17AAG (C) or 17DMAG (D) for 72 hours prior to determination of virus production. Data represent the mean 50% tissue culture infective dose per mL (TCID50/mL) and SEM of ≥3 experiments. (E) Antiviral activity of Hsp90 inhibitors against clinical isolates of RSV in HEp-2 cells. Cells infected with clinical isolates 76 and 90 were treated with the indicated concentrations of the Hsp90 inhibitors 17AAG and 17DMAG, respectively. * p

    Journal: PLoS ONE

    Article Title: Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus

    doi: 10.1371/journal.pone.0056762

    Figure Lengend Snippet: Hsp90 inhibitors have anti-RSV activity at non-toxic concentrations. (A–B) Toxicity of 17AAG (A) or 17DMAG (B) in HEp-2 cells was assessed after 72 hours using the Alamar blue cytotoxicity assay. (C–D) Hsp90 inhibitors reduce RSV replication. HEp-2 cells infected with RSV-A2 at an MOI of 0.01 were treated with the indicated concentrations of 17AAG (C) or 17DMAG (D) for 72 hours prior to determination of virus production. Data represent the mean 50% tissue culture infective dose per mL (TCID50/mL) and SEM of ≥3 experiments. (E) Antiviral activity of Hsp90 inhibitors against clinical isolates of RSV in HEp-2 cells. Cells infected with clinical isolates 76 and 90 were treated with the indicated concentrations of the Hsp90 inhibitors 17AAG and 17DMAG, respectively. * p

    Article Snippet: Cells, viruses, and reagents HEp-2 cells (CCL-23) were purchased from ATCC and maintained in DMEM/F12 (Invitrogen), 10% FCS, and antibiotics at 37°C.

    Techniques: Activity Assay, Cytotoxicity Assay, Infection

    Mechanism of action underlying the antiviral activity of Hsp90 inhibitors against RSV. (A) Hsp90 inhibitors do not act via inhibition of early steps in the RSV cycle, and significantly reduce RSV replication even when added 24 hours post infection. HEp-2 cells were infected with RSV-A2 at an MOI of 1. 17AAG (0.1 µM) was added at 0, 24, or 48 hours post infection and virus production measured 72 hours post infection. Data represents mean and SEM of 4 experiments. (B) The antiviral activity of Hsp90 inhibitors is independent of direct effects on virus infectivity. RSV was incubated with 1 µM 17AAG or DMSO as a control for 90 minutes at 4°C prior to infectivity determination using endpoint titration. Data represent the mean TCID50/mL and SEM of 3 experiments.

    Journal: PLoS ONE

    Article Title: Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus

    doi: 10.1371/journal.pone.0056762

    Figure Lengend Snippet: Mechanism of action underlying the antiviral activity of Hsp90 inhibitors against RSV. (A) Hsp90 inhibitors do not act via inhibition of early steps in the RSV cycle, and significantly reduce RSV replication even when added 24 hours post infection. HEp-2 cells were infected with RSV-A2 at an MOI of 1. 17AAG (0.1 µM) was added at 0, 24, or 48 hours post infection and virus production measured 72 hours post infection. Data represents mean and SEM of 4 experiments. (B) The antiviral activity of Hsp90 inhibitors is independent of direct effects on virus infectivity. RSV was incubated with 1 µM 17AAG or DMSO as a control for 90 minutes at 4°C prior to infectivity determination using endpoint titration. Data represent the mean TCID50/mL and SEM of 3 experiments.

    Article Snippet: Cells, viruses, and reagents HEp-2 cells (CCL-23) were purchased from ATCC and maintained in DMEM/F12 (Invitrogen), 10% FCS, and antibiotics at 37°C.

    Techniques: Activity Assay, Activated Clotting Time Assay, Inhibition, Infection, Incubation, Titration

    The RNA polymerase of RSV is a likely Hsp90 client protein. (A) Hsp90 inhibitors destabilize a viral protein of 250 kDa, corresponding to the size of the RSV polymerase, the L protein. Control or RSV-infected HEp-2 cells were treated with 17AAG or DMSO and labeled with 35 S-methionine/cysteine. Viral proteins were immunoprecipitated, separated by 12% SDS-PAGE, and visualized by autoradiography. The Hsp90 inhibitor 17AAG specifically reduced the intensity of the 250 kDa protein by ∼60%. (B) Hsp90 inhibition leads to proteasomal degradation of the viral L protein. Control or RSV-infected HEp-2 cells were treated with 17AAG, 17AAG and the proteasome inhibitor MG132, or DMSO and labeled with 35 S-methionine/cysteine. The reduction of L protein caused by 17AAG is abrogated by MG132. Viral proteins were immunoprecipitated, separated by 12% SDS-PAGE, and visualized by autoradiography. Densitometry analysis showed a band intensity of 40% and 75% for 17AAG treatment and the combined 17AAG/MG132 treatment, respectively, relative to control conditions. ** p

    Journal: PLoS ONE

    Article Title: Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus

    doi: 10.1371/journal.pone.0056762

    Figure Lengend Snippet: The RNA polymerase of RSV is a likely Hsp90 client protein. (A) Hsp90 inhibitors destabilize a viral protein of 250 kDa, corresponding to the size of the RSV polymerase, the L protein. Control or RSV-infected HEp-2 cells were treated with 17AAG or DMSO and labeled with 35 S-methionine/cysteine. Viral proteins were immunoprecipitated, separated by 12% SDS-PAGE, and visualized by autoradiography. The Hsp90 inhibitor 17AAG specifically reduced the intensity of the 250 kDa protein by ∼60%. (B) Hsp90 inhibition leads to proteasomal degradation of the viral L protein. Control or RSV-infected HEp-2 cells were treated with 17AAG, 17AAG and the proteasome inhibitor MG132, or DMSO and labeled with 35 S-methionine/cysteine. The reduction of L protein caused by 17AAG is abrogated by MG132. Viral proteins were immunoprecipitated, separated by 12% SDS-PAGE, and visualized by autoradiography. Densitometry analysis showed a band intensity of 40% and 75% for 17AAG treatment and the combined 17AAG/MG132 treatment, respectively, relative to control conditions. ** p

    Article Snippet: Cells, viruses, and reagents HEp-2 cells (CCL-23) were purchased from ATCC and maintained in DMEM/F12 (Invitrogen), 10% FCS, and antibiotics at 37°C.

    Techniques: Infection, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Inhibition

    Extensive growth of RSV in the presence of Hsp90 inhibitors does not select for the emergence of drug resistance. Antiviral activity of 17AAG against an RSV viral population obtained following 15 passages in the presence of 17AAG and a control RSV viral population. HEp-2 cells were infected with the 17AAG passaged viral population or a control viral population and treated with the indicated concentration of 17AAG for 72 hours. Virus production was then assayed by endpoint dilution. Data indicate the mean TCID50/mL and SEM of ≥2 experiments.

    Journal: PLoS ONE

    Article Title: Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus

    doi: 10.1371/journal.pone.0056762

    Figure Lengend Snippet: Extensive growth of RSV in the presence of Hsp90 inhibitors does not select for the emergence of drug resistance. Antiviral activity of 17AAG against an RSV viral population obtained following 15 passages in the presence of 17AAG and a control RSV viral population. HEp-2 cells were infected with the 17AAG passaged viral population or a control viral population and treated with the indicated concentration of 17AAG for 72 hours. Virus production was then assayed by endpoint dilution. Data indicate the mean TCID50/mL and SEM of ≥2 experiments.

    Article Snippet: Cells, viruses, and reagents HEp-2 cells (CCL-23) were purchased from ATCC and maintained in DMEM/F12 (Invitrogen), 10% FCS, and antibiotics at 37°C.

    Techniques: Activity Assay, Infection, Concentration Assay

    Effect of the time-of-addition assay of the Adenanthera pavonina sulfated polysaccharide (SPLS Ap ) on poliovirus type 1 (PV-1) in HEp-2 cells by plaque reduction assay. The percentage of viral inhibition (%VI) is expressed as mean ± SD of triplicate independent experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Antiviral Activity of Sulfated Polysaccharide of Adenanthera pavonina against Poliovirus in HEp-2 Cells

    doi: 10.1155/2014/712634

    Figure Lengend Snippet: Effect of the time-of-addition assay of the Adenanthera pavonina sulfated polysaccharide (SPLS Ap ) on poliovirus type 1 (PV-1) in HEp-2 cells by plaque reduction assay. The percentage of viral inhibition (%VI) is expressed as mean ± SD of triplicate independent experiments.

    Article Snippet: Cells and Virus HEp-2 cells (epithelial human larynx carcinoma cells, ATCC CCL-23) were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (∗Life Technology Corp., USA), and treated with 100 μ g/mL streptomycin (∗), 100 IU/mL of penicillin (Novafarma Indústria Farmacêutica, BR), and 2.5 μ g/mL of amphotericin B (Meizler Biopharma, BR).

    Techniques: Inhibition

    The effect of the Adenanthera pavonina sulfated polysaccharide (SPLS Ap ) on poliovirus type 1 (PV-1) particle (virucidal activity) and in virus adsorption on HEp-2 cells by plaque reduction assay. The percentage of viral inhibition (%VI) is expressed as the mean ± SD of triplicate independent experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Antiviral Activity of Sulfated Polysaccharide of Adenanthera pavonina against Poliovirus in HEp-2 Cells

    doi: 10.1155/2014/712634

    Figure Lengend Snippet: The effect of the Adenanthera pavonina sulfated polysaccharide (SPLS Ap ) on poliovirus type 1 (PV-1) particle (virucidal activity) and in virus adsorption on HEp-2 cells by plaque reduction assay. The percentage of viral inhibition (%VI) is expressed as the mean ± SD of triplicate independent experiments.

    Article Snippet: Cells and Virus HEp-2 cells (epithelial human larynx carcinoma cells, ATCC CCL-23) were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (∗Life Technology Corp., USA), and treated with 100 μ g/mL streptomycin (∗), 100 IU/mL of penicillin (Novafarma Indústria Farmacêutica, BR), and 2.5 μ g/mL of amphotericin B (Meizler Biopharma, BR).

    Techniques: Activity Assay, Adsorption, Inhibition

    Characteristic staining patterns of anti-lysosomes/endosomes ( a ), anti-endoplasmatic reticulum ( b , c ), anti-Golgi apparatus ( d ) for confocal microscopy on home-made HEp-2 substrate

    Journal: Auto-Immunity Highlights

    Article Title: Characterization for anti-cytoplasmic antibodies specificity by morphological and molecular techniques

    doi: 10.1007/s13317-012-0033-4

    Figure Lengend Snippet: Characteristic staining patterns of anti-lysosomes/endosomes ( a ), anti-endoplasmatic reticulum ( b , c ), anti-Golgi apparatus ( d ) for confocal microscopy on home-made HEp-2 substrate

    Article Snippet: For confirmatory techniques, human HEp-2 cells (carcinoma tissue from the larynx, ATCC) were cultured into 75 cm2 home-made flasks in Dulbecco’s minimal essential medium supplemented with 10 % fetal bovine serum, 1 % glutamine, 100 units penicillin and streptomycin (Celbio) in a 5 % CO2 humidified atmosphere.

    Techniques: Staining, Confocal Microscopy

    Immunoprecipitation assay and related molecular weights (kDa) with endoplasmatic reticulum autoantibodies, Calnexin-marker antibody (protein band runs around 90 kDa) and PDI marker antibody (Pancreas-specific protein disulfide isomerase) from HEp-2 cell lysate

    Journal: Auto-Immunity Highlights

    Article Title: Characterization for anti-cytoplasmic antibodies specificity by morphological and molecular techniques

    doi: 10.1007/s13317-012-0033-4

    Figure Lengend Snippet: Immunoprecipitation assay and related molecular weights (kDa) with endoplasmatic reticulum autoantibodies, Calnexin-marker antibody (protein band runs around 90 kDa) and PDI marker antibody (Pancreas-specific protein disulfide isomerase) from HEp-2 cell lysate

    Article Snippet: For confirmatory techniques, human HEp-2 cells (carcinoma tissue from the larynx, ATCC) were cultured into 75 cm2 home-made flasks in Dulbecco’s minimal essential medium supplemented with 10 % fetal bovine serum, 1 % glutamine, 100 units penicillin and streptomycin (Celbio) in a 5 % CO2 humidified atmosphere.

    Techniques: Immunoprecipitation, Marker

    Characteristic staining patterns of anti-Golgi apparatus autoantibodies ( a , b ), anti-lysosomes/endosomes ( c ) and anti-endoplasmatic reticulum ( d ) on a HEp-2 commercial substrate

    Journal: Auto-Immunity Highlights

    Article Title: Characterization for anti-cytoplasmic antibodies specificity by morphological and molecular techniques

    doi: 10.1007/s13317-012-0033-4

    Figure Lengend Snippet: Characteristic staining patterns of anti-Golgi apparatus autoantibodies ( a , b ), anti-lysosomes/endosomes ( c ) and anti-endoplasmatic reticulum ( d ) on a HEp-2 commercial substrate

    Article Snippet: For confirmatory techniques, human HEp-2 cells (carcinoma tissue from the larynx, ATCC) were cultured into 75 cm2 home-made flasks in Dulbecco’s minimal essential medium supplemented with 10 % fetal bovine serum, 1 % glutamine, 100 units penicillin and streptomycin (Celbio) in a 5 % CO2 humidified atmosphere.

    Techniques: Staining

    Immunoprecipitation assay and related molecular weights with lysosomes/endosomes autoantibodies and LC3B marker antibody from HEp-2 cell lysate

    Journal: Auto-Immunity Highlights

    Article Title: Characterization for anti-cytoplasmic antibodies specificity by morphological and molecular techniques

    doi: 10.1007/s13317-012-0033-4

    Figure Lengend Snippet: Immunoprecipitation assay and related molecular weights with lysosomes/endosomes autoantibodies and LC3B marker antibody from HEp-2 cell lysate

    Article Snippet: For confirmatory techniques, human HEp-2 cells (carcinoma tissue from the larynx, ATCC) were cultured into 75 cm2 home-made flasks in Dulbecco’s minimal essential medium supplemented with 10 % fetal bovine serum, 1 % glutamine, 100 units penicillin and streptomycin (Celbio) in a 5 % CO2 humidified atmosphere.

    Techniques: Immunoprecipitation, Marker

    Polyspecificity analysis of MinVRC01 and Min12A21. ( A ) HEp-2 cell staining assay, for which 4E10 is a positive control while Humira, 10E8, and undiluted human serum

    Journal: PLoS Pathogens

    Article Title: Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design

    doi: 10.1371/journal.ppat.1005815

    Figure Lengend Snippet: Polyspecificity analysis of MinVRC01 and Min12A21. ( A ) HEp-2 cell staining assay, for which 4E10 is a positive control while Humira, 10E8, and undiluted human serum "Negative control" are negative controls. ( B ) Polyspecificity reagent (PSR) binding assay measuring binding to preparations of solubilized membrane proteins and cytosolic proteins from CHO cells. Here, 4E10 and 2F5 are positive controls, while Humira, 10E8, PG9 and 40 HPV-vaccine induced human mAbs are negative controls. Error bars reflect mean ± standard deviation with N = 40.

    Article Snippet: These Aesku slides use optimally fixed human epithelial (HEp-2) cells (ATCC) as substrate and affinity purified, FITC-conjugated goat anti-human IgG for the detection.

    Techniques: Staining, Positive Control, Negative Control, Binding Assay, Standard Deviation