Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route
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{"title"=>"Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route", "type"=>"journal", "authors"=>[{"first_name"=>"Kepa B.", "last_name"=>"Uribe", "scopus_author_id"=>"40561765500"}, {"first_name"=>"César", "last_name"=>"Martín", "scopus_author_id"=>"7405843847"}, {"first_name"=>"Aitor", "last_name"=>"Etxebarria", "scopus_author_id"=>"24173037500"}, {"first_name"=>"David", "last_name"=>"González-Bullón", "scopus_author_id"=>"55879473100"}, {"first_name"=>"Geraxane", "last_name"=>"Gómez-Bilbao", "scopus_author_id"=>"35279644500"}, {"first_name"=>"Helena", "last_name"=>"Ostolaza", "scopus_author_id"=>"6603076490"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203", "issn"=>"19326203", "pui"=>"369813422", "sgr"=>"84884166360", "pmid"=>"24058533", "scopus"=>"2-s2.0-84884166360", "doi"=>"10.1371/journal.pone.0074248"}, "id"=>"4bf3c1c8-24f5-3285-aeb4-90a6a56652f6", "abstract"=>"Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+) influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.", "link"=>"http://www.mendeley.com/research/ca2-influx-tyrosine-kinases-trigger-bordetella-adenylate-cyclase-toxin-act-endocytosis-cell-physiolo", "reader_count"=>26, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>3, "Student > Master"=>2, "Student > Bachelor"=>9, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>3, "Student > Postgraduate"=>3, "Student > Master"=>2, "Student > Bachelor"=>9, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>14, "Medicine and Dentistry"=>1, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>14}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Spain"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1204335"], "description"=>"<p>CHO-K1 cells stably transfected to express CD11b and CD18 (CR3<sup>+</sup>CHO-K1 cells) and mock control cells were used to assess internalisation of ACT and of its receptor, the integrin CD11b/CD18 or CR3, as well as to test the effect of several inhibitors into the ACT-induced internalisation processes. The concentrations of the different compounds, used in these experiments, are identical to those used in the former experiments. (<b>A</b>) Internalisation of the CD11b/CD18 integrin by the CR3<sup>+</sup>CHO-K1 cells. (<b>B</b>) ACT internalisation. Internalisation was determined by FACS as described in <i>Materials and Methods</i>. Data shown are the mean ± SD of at least three independent experiments, with **p<0.025 and ***p<0.001.</p>", "links"=>[], "tags"=>["receptor", "internalised"], "article_id"=>799323, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g007", "stats"=>{"downloads"=>0, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ACT_and_its_CD11b_CD18_receptor_are_internalised_by_CR3_CHO_K1_cells_/799323", "title"=>"ACT and its CD11b/CD18 receptor are internalised by CR3<sup>+</sup>CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204329"], "description"=>"<p>Cells were transfected with anti-Cav-1 siRNA or with a non-effective scrambled vector (mock cells). Then, silenced or mock cells were incubated with 35 nM ACT at 37°C for 10 min and surface staining of ACT followed in a flow cytometer. Internalisation of ACT was determined by FACS as described in <i>Materials and Methods</i> (<b>A</b>). Western blotting detection of caveolin-1 protein expression in CHO-K1 cells after transfection with the siRNA molecules (<b>B</b>). Cells were transfected with anti-Clathrin siRNA or with a non-effective scrambled vector (mock cells). Then, silenced or mock cells were incubated with 35 nM ACT at 37°C for 10 min and surface staining of ACT followed in a flow cytometer. Internalisation of ACT was determined by FACS as described in <i>Materials and Methods</i> (<b>C</b>). Western blotting detection of clathrin expression in CHO-K1 cells after transfection with the siRNA molecules (<b>D</b>). The procedure was performed as described in <i>Materials and Methods</i>. Equal loading of protein was confirmed in each blot by membrane stripping and further incubation with antibodies to visualize cytosolic GAPDH protein (Lower panel of the figure). The extent of protein silencing was determined by quantitative densitometric analysis. Data shown in the left-hand panels of the figure are the mean ± SD of at least three independent experiments, with *p<0.001.</p>", "links"=>[], "tags"=>["caveolin-1", "internalisation", "cho-k1"], "article_id"=>799317, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g005", "stats"=>{"downloads"=>5, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knock_down_of_caveolin_1_and_and_clathrin_and_its_effect_on_the_ACT_internalisation_into_CHO_K1_cells_/799317", "title"=>"Knock down of caveolin-1 and and clathrin, and its effect on the ACT internalisation into CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204347"], "description"=>"<p>(<b>A</b>) Pre-incubation of the different cells with nifedipine (10 µM), which was previously shown to effectively block the ACT-induced Ca<sup>2+</sup> influx <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074248#pone.0074248-Martn1\" target=\"_blank\">[18]</a>, very importantly decreased the toxin uptake by either CHO-K1 cells, as CR3<sup>+</sup>CHO-K1 cells or by J774A.1 and CR3<b><sup>−</sup></b>J774A.1 cells, hence suggesting that Ca<sup>2+</sup> influx induced by ACT is a common master denominator in the different endocytosis routes used by the different cells to take the toxin. Cells were preincubated 30 min at 37°C, then 35 nM ACT was added and cells further incubated for 10 min. Internalisation was determined by FACS as described in <i>Materials and Methods</i>. Data shown are the mean ± SD of at least three independent experiments, with *p<0.001. (<b>B</b>) The intracellular calcium level was measured using FURA-2, as described in the <i>Methods</i> section, in the different cell lines, in presence or absence of nifedipine. Data shown are the mean ± SD of at least three independent experiments, with *p<0.001.</p>", "links"=>[], "tags"=>["influx", "triggers", "toxin", "uptake"], "article_id"=>799331, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g011", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ACT_induced_Ca_2_influx_triggers_the_toxin_uptake_by_the_different_target_cells_/799331", "title"=>"ACT-induced Ca<sup>2+</sup> influx triggers the toxin uptake by the different target cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204328"], "description"=>"<p>(A) Western blot analysis of sucrose density gradient fractions obtained by centrifugation of Triton X-100-solubilized CHO-K1 cells treated with 35 nM ACT showed that the toxin was recovered from high-buoyant density fractions of the gradient, and that it co-localized with caveolin-1 and flotillin. The procedure was performed as described in <i>Materials and Methods</i>. A representative experiment from three independently performed assays is shown. (B) Confocal microscopy analysis of the localization of ACT and Cav-1 in CHO-K1 cells. Cells were incubated for 10 min with the toxin, washed three times, fixed and permeabilized to analyze ACT and Cav-1 by immunohistochemistry. A representative image is shown.</p>", "links"=>[], "tags"=>["blot", "sucrose", "gradient", "fractions", "confocal", "microscopy", "images", "localization", "act-treated", "cho-k1"], "article_id"=>799316, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g004", "stats"=>{"downloads"=>1, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Western_blot_analysis_of_sucrose_density_gradient_fractions_and_confocal_microscopy_images_showing_ACT_localization_in_ACT_treated_CHO_K1_cells_/799316", "title"=>"Western blot analysis of sucrose density gradient fractions and confocal microscopy images showing ACT localization in ACT-treated CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204363", "https://ndownloader.figshare.com/files/1204364", "https://ndownloader.figshare.com/files/1204365", "https://ndownloader.figshare.com/files/1204367", "https://ndownloader.figshare.com/files/1204368", "https://ndownloader.figshare.com/files/1204369"], "description"=>"<div><p>Humans infected with <i>Bordetella pertussis</i>, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca<sup>2+</sup> influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.</p></div>", "links"=>[], "tags"=>["influx", "tyrosine", "kinases", "adenylate", "cyclase", "toxin", "physiology", "integrin", "determinants"], "article_id"=>799345, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0074248.s001", "https://dx.doi.org/10.1371/journal.pone.0074248.s002", "https://dx.doi.org/10.1371/journal.pone.0074248.s003", "https://dx.doi.org/10.1371/journal.pone.0074248.s004", "https://dx.doi.org/10.1371/journal.pone.0074248.s005", "https://dx.doi.org/10.1371/journal.pone.0074248.s006"], "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Ca_2_Influx_and_Tyrosine_Kinases_Trigger_Bordetella_Adenylate_Cyclase_Toxin_ACT_Endocytosis_Cell_Physiology_and_Expression_of_the_CD11b_CD18_Integrin_Major_Determinants_of_the_Entry_Route/799345", "title"=>"Ca<sup>2+</sup> Influx and Tyrosine Kinases Trigger <i>Bordetella</i> Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204346"], "description"=>"<p>J774A.1 cells were pre-incubated for 30 min at 37°C with PP2 to inhibit Src Tyr Kinases and PP3 as negative control for PP2. Then ACT was added at a final concentration of 35 nM, and cells further incubated at 37°C for 10 min. Internalisation of ACT and CD11b was assessed by FACS and expressed as described in <i>Materials and Methods</i>. Data shown are the mean ± SD of at least three independent experiments, with *p<0.001.</p>", "links"=>[], "tags"=>["receptor", "mediated", "src", "tyr", "kinases"], "article_id"=>799330, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g010", "stats"=>{"downloads"=>2, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Endocytosis_of_ACT_and_its_CD11b_CD18_receptor_is_mediated_by_Src_Tyr_kinases_in_J774A_1_cells_/799330", "title"=>"Endocytosis of ACT and its CD11b/CD18 receptor is mediated by Src Tyr kinases in J774A.1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204324"], "description"=>"<p>Addition of ACT (35 nM) to CHO-K1 cells results in time-dependent internalisation of the toxin, as determined by flow cytometry and described in <i>Materials and Methods</i>. Geometric mean fluorescence intensities (GMFI) were used to show ACT internalisation. The data shown are the mean ± SD of at least three independent experiments, with *p<0.05, **p<0.025 and ***p<0.001.</p>", "links"=>[], "tags"=>["internalisation", "cho-k1"], "article_id"=>799312, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g001", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Kinetics_of_ACT_internalisation_into_CHO_K1_cells_/799312", "title"=>"Kinetics of ACT internalisation into CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204341"], "description"=>"<p>A knockdown experiment, in which expression of Cav-1 and clathrin was silenced by the corresponding siRNA molecules, was performed in the CR3<sup>+</sup>CHO-K1 cells. Then, the silenced or control mock cells were incubated with ACT at 37°C and surface staining of ACT followed in a flow cytometer. (<b>A</b>) Extent of CD11b internalisation upon silencing the expression of Cav-1 or clathrin, (<b>B</b>) Extent of ACT internalisation upon silencing the expression of Cav-1 or clathrin, Internalisation was determined by FACS as described in <i>Materials and Methods</i>. (<b>C</b>) Western blotting detection of clathrin protein expression in CR3<sup>+</sup>CHO-K1 cells after transfection with anti-siRNA. (<b>D</b>) Western blotting detection of caveolin-1 protein expression in CR3<sup>+</sup>CHO-K1 cells after transfection with anti-cav-1 siRNA molecules. Extent of protein silencing was determined as described in the legend of Fig. 5. Data shown in the upper panels are the mean ± SD of at least three independent experiments, with **p<0.025, ***p<0.001.</p>", "links"=>[], "tags"=>["caveolin-1", "internalisation"], "article_id"=>799325, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g008", "stats"=>{"downloads"=>1, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knock_down_of_caveolin_1_or_clathrin_and_its_effect_on_the_ACT_internalisation_into_the_CR3_CHO_K1_cells_/799325", "title"=>"Knock down of caveolin-1 or clathrin, and its effect on the ACT internalisation into the CR3<sup>+</sup>CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204331"], "description"=>"<p>Phosphorylation of Cav-1 in Tyr 14 was detected by Western blot of cell lysates incubated with the toxin (35 nM) for 5 min at 37°C (<b>A</b>). Caveolin-1 phosphorylation is significantly raised in the ACT-treated cells compared to control cells. ACT treatment raised about three times the Cav-1 phosphorylation in target cells. In addition, incubation of cells with PP2 (5 nM) significantly decreased the degree of Cav-1 phosphorylation, being similar to control cells. Incubation with PP3, the negative control of PP2, did not affect the Cav-1 phosphorylation induced by ACT treatment. Equal loading of protein was confirmed using specific antibodies to cytosolic GAPDH protein, after membrane stripping. Intensity of the bands was determined by scanning densitometry. (<b>B</b>) Effect of PP2 and PP3 on the ACT internalisation extent. CHO-K1 cells were pre-incubated for 30 min at 37°C with PP2 (5 nM) or PP3 (2,7 µM). Then cells were incubated with 35 nM ACT at 37°C for 10 min. Then, the surface staining of ACT was measured using a flow cytometer. Internalisation was assessed by FACS and expressed as described in <i>Materials and Methods</i>. Data shown in the lower panel are the mean ± SD of at least three independent experiments, with **p<0.025.</p>", "links"=>[], "tags"=>["cav-1", "act-treated", "cho-k1"], "article_id"=>799319, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g006", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phosphorylation_of_Cav_1_in_ACT_treated_CHO_K1_cells_/799319", "title"=>"Phosphorylation of Cav-1 in ACT-treated CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204327"], "description"=>"<p>CHO-K1 cells were pre-incubated for 30 min at 37°C with three different cholesterol depleting agents, methyl-β-cyclodextrin (10 mM), nystatin (0.25 µg/ml) and filipin (0.5 µg/ml). Cells were incubated with 35 nM ACT at 37°C for 10 min. Then, the surface staining of ACT was measured using a flow cytometer. Internalisation was assessed by FACS and expressed as described in <i>Materials and Methods</i>. Data shown are the mean ± SD of at least three independent experiments, with ***p<0.001.</p>", "links"=>[], "tags"=>["depleting", "agents", "internalisation", "cho-k1"], "article_id"=>799315, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g003", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_cholesterol_depleting_agents_on_the_internalisation_of_ACT_into_CHO_K1_cells_/799315", "title"=>"Effect of cholesterol depleting agents on the internalisation of ACT into CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204348"], "description"=>"<p>CHO cells were treated with 35-CR3 cells with 5 nM ACT for different incubation times (5, 10, 30, 60 and 120 min), then washed, trypsinized and incubated with propidium iodide for 4 min. PI uptake by the different cells was determined by flow cytometry as described in Materials and Methods section. Population of viable cells which show PI staining were gated in a FSH <i>vs</i> PI density plot, and propidium iodide positive cells were enumerated; data are displayed as fold activation over untreated controls with data-points representing mean values of at least three independent experiments ± SEM of at least three independent experiments, with **p<0.025, ***p<0.001.</p>", "links"=>[], "tags"=>["propidium", "iodide", "act-treated", "cho"], "article_id"=>799332, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g012", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Uptake_of_propidium_iodide_PI_by_ACT_treated_CHO_and_CR3_CHO_K1_cells_/799332", "title"=>"Uptake of propidium iodide (PI) by ACT-treated CHO and CR3<sup>+</sup>CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204345"], "description"=>"<p>J774A.1 cells in which CD11b expression was silenced by an anti-CD11b shRNA were used to assess ACT internalisation into these CR3<b><sup>−</sup></b> macrophages, as well as to test the effect of several inhibitors into this internalisation. The concentrations of the different compounds, used in these experiments, are identical to those used in the former experiments. Internalisation was determined by FACS as described in <i>Materials and Methods</i>. Data shown are the mean ± SD of at least three independent experiments, with ***p<0.001.</p>", "links"=>[], "tags"=>["endocytosed", "pathway"], "article_id"=>799329, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g009", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ACT_is_endocytosed_by_clathrin_dependent_but_receptor_independent_pathway_in_CR3_8722_J774A_1_cells_/799329", "title"=>"ACT is endocytosed by clathrin-dependent, but receptor-independent, pathway in CR3<sup>−</sup>J774A.1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/1204326"], "description"=>"<p>(<b>A</b>) CHO-K1 cells were pre-incubated for 30 min at 37°C with several known inhibitors of endocytosis routes, at the following concentrations: sucrose (450 mM), chlorpromazine (5 µg/ml), DMA (200 µM), nocodazole (20 µM), cytochalasin D (10 µM). Then toxin was added at a final concentration of 35 nM, and cells further incubated at 37°C for 10 min. Then, the surface staining of ACT was measured using a flow cytometer. (<b>B</b>) Transferrin uptake by the CHO-K1 cells was used as a control assay to be sure that the clathrin-dependent endocytosis was fully functional, and that ACT did not interfere with such internalisation. Internalisation was assessed by FACS and expressed as described in <i>Materials and Methods</i>. Data shown are the mean ± SD of at least three independent experiments, with ***p<0.001.</p>", "links"=>[], "tags"=>["inhibitors", "internalisation", "cho-k1"], "article_id"=>799314, "categories"=>["Biological Sciences"], "users"=>["Kepa B. Uribe", "César Martín", "Aitor Etxebarria", "David González-Bullón", "Geraxane Gómez-Bilbao", "Helena Ostolaza"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0074248.g002", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_several_inhibitors_on_the_internalisation_of_ACT_into_CHO_K1_cells_/799314", "title"=>"Effect of several inhibitors on the internalisation of ACT into CHO-K1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-13 02:05:05"}

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Relative Metric

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