Journal: PLoS Pathogens

Article Title: cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

doi: 10.1371/journal.ppat.1002664

Figure Lengend Snippet: (A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (left panel) or GFP-Rap2b ΔAAX (right panel). Twenty-four hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) in full nutrient media, or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of three independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown. * p <0.05, ** p <0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments. (C) CHO cells were transfected with GFP-Rap2b wt or GFP-Rap2b ΔAAX and incubated with complete medium without (Ctr) or with 10 µg/ml of α-hemolysin (Hla) for 4 h, with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h in the absence or presence of bafilomycin A1. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments. (D) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program.

Article Snippet: The anti-myc antibody, the anti-Rap2b antibody, and Rap2b siRNA were purchased from Santa Cruz Biotechnology (Buenos Aires, Argentina).

Techniques: Transfection, Incubation, Control, Confocal Microscopy, Western Blot, Labeling

Journal: PLoS Pathogens

Article Title: cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

doi: 10.1371/journal.ppat.1002664

Figure Lengend Snippet: (A) HeLa cells were cotransfected with Rap2b siRNA and GFP-LC3 (right panel) or irrelevant siRNA and GFP-LC3 (left panel). Forty-eight hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions. Data correspond to two independent experiments ( n = 50 cells/condition). (C) Upper panel: The knockdown of Rap2b was determined by Western blot as indicated in . Lower panel: The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p <0.05 (paired Student's t-test). (D) HeLa cells were cotransfected with GFP-LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence (Ctr) or presence of 10 µg/ml of α-hemolysin (Hla), with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments. (E) The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p <0.05 (paired Student's t-test).

Article Snippet: The anti-myc antibody, the anti-Rap2b antibody, and Rap2b siRNA were purchased from Santa Cruz Biotechnology (Buenos Aires, Argentina).

Techniques: Transfection, Incubation, Control, Confocal Microscopy, Knockdown, Western Blot, Labeling

Journal: PLoS Pathogens

Article Title: cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

doi: 10.1371/journal.ppat.1002664

Figure Lengend Snippet: (A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.

Article Snippet: The anti-myc antibody, the anti-Rap2b antibody, and Rap2b siRNA were purchased from Santa Cruz Biotechnology (Buenos Aires, Argentina).

Techniques: Transfection, Incubation, Control, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

doi: 10.1371/journal.ppat.1002664

Figure Lengend Snippet: (A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (upper panel) or GFP-Rap2b ΔAAX (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in and immediately visualized by confocal microscopy. These data are representative of three independent experiments. (B) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection. * p <0.05, *** p <0.001 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments. (C) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection. ** p <0.01 (paired Student's t-test, n = 30 cells/condition). These data are representative of three independent experiments.

Article Snippet: The anti-myc antibody, the anti-Rap2b antibody, and Rap2b siRNA were purchased from Santa Cruz Biotechnology (Buenos Aires, Argentina).

Techniques: Transfection, Infection, Mutagenesis, Expressing, Plasmid Preparation, Bacteria, Labeling, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

doi: 10.1371/journal.ppat.1002664

Figure Lengend Snippet: (A) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel. These data are representative of two independent experiments. (B) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Cells were disrupted and whole cell lysates were subjected to pull-down assays using GST-Ral-GDS-RBD-sepharose. The levels of GTP-bound Rap2b were determined as was described in by Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel. * p <0.05 (paired Student's t-test). These data are representative of two independent experiments.

Article Snippet: The anti-myc antibody, the anti-Rap2b antibody, and Rap2b siRNA were purchased from Santa Cruz Biotechnology (Buenos Aires, Argentina).

Techniques: Incubation, Infection, Mutagenesis, Western Blot, Labeling

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Platelets promote human macrophages-mediated macropinocytosis of Clostridioides difficile

doi: 10.3389/fcimb.2023.1252509

Figure Lengend Snippet: Interaction of platelets with C. difficile , monocytes and macrophages. (A–C) Washed Platelets (WP) from Healthy Donors (HD) were incubated with heat-inactivated C. difficile coupled to FITC ( CD H FITC) at ratios 1:1 and 1:10 for 2h. CD H FITC-WP complexes were detected by flow cytometry (A) and fluorescence microscopy (B, C) using anti-CD61 PE antibody. (A, B) are representative experiments. (B) Images on the right are magnifications corresponding to white boxes on the left. (C) Quantification of CD H FITC-associated platelets from fluorescent micrographs is shown. (D) WP from HD were incubated with live CD160 and live NAP1/BI/027 C. difficile strains coupled to FITC and with CD H FITC plus NAP1/BI/027 secretome ( CD H FITC+Sec) for 2h. In all cases, WP:bacteria ratios of 1:1 and 1:10 were tested and C. difficile -WP complexes were detected by flow cytometry. Representative histograms are shown. (E) Whole peripheral blood from HD was cultured in the presence or absence of CD H for 2h. Monocytes (Mo)-WP complexes were measured by flow cytometry using anti-CD14 Alexa Fluor 647 and anti-CD61 PE antibodies. (F) Macrophages (MΦs) and WP from HD were co-cultured in the presence or absence of CD H for 1h. MΦs-WP complexes were assessed by confocal microscopy after fixation, permeabilization and staining. The white boxes correspond to the magnified images on the right (representative experiment). White arrowheads point WP inside MΦs. DAPI (nuclei) is shown in blue, CD61 (platelets) in red and CD14 (macrophages) in purple. DIC: differential interference contrast. Scale bar: 10 μm (A, C, F) correspond to three individual donors in three independent experiments, (D) corresponds to four independent experiments, (E) corresponds to five individual donors in five independent experiments. (C) Bars represent the mean ± SEM. (E) Each dot corresponds to an individual healthy donor. (C) Mann-Whitney test, (E) Wilcoxon test. *p<0.05, ****p<0.0001.

Article Snippet: In vitro infection of cells was performed with vegetative live non-toxigenic CD160 (NR-43516, BEI Resources) or hypervirulent NAP1/BI/027 (Sanitary Bacteriology Service INEI-ANLIS, Dr. Carlos G. Malbrán, Argentina) C. difficile strains.

Techniques: Incubation, Flow Cytometry, Fluorescence, Microscopy, Bacteria, Cell Culture, Confocal Microscopy, Staining, MANN-WHITNEY

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Platelets promote human macrophages-mediated macropinocytosis of Clostridioides difficile

doi: 10.3389/fcimb.2023.1252509

Figure Lengend Snippet: Evaluation of endocytic pathways against C. difficile . A) Macrophages (MΦs) from Healthy Donors (HD) were cultured with endocytosis inhibitors (Citochalasin B (Cit B, actin polymerization inhibitor), Citochalasin D (Cit D, inhibitor of actin polymerization by ATP hydrolysis), Colchicine (Colch, microtubules polymerization inhibitor), Vincristine (Vincr, microtubules polymerization inhibitor), Nystatin (Nist, inhibitor of caveolae/lipid mediated endocytosis), Bafilomycin (Bafilo, autophagy inhibitor) or Amiloride (Amilo, macropinocytosis inhibitor)). After 30min, heat-inactivated C. difficile coupled to FITC ( CD H FITC) was added for 1h. CD H FITC internalization was assessed by flow cytometry (A, B) and confocal microscopy (C) . (A) Percentage of FITC positive cells (uptake). (B) Representative histograms. (C) Confocal micrographs of untreated (-) or Amiloride treated (Amilo) MΦs obtained after cell fixation, staining with anti-CD14 Alexa Fluor 647 antibody (macrophages) and DAPI (nuclei) counterstaining. The white boxes correspond to the magnified images on the right. White arrowheads point internalized CD H FITC. (D) MΦs were stimulated with CD H FITC in the presence or absence of Washed Platelets (WP) (ratio 1:10:100) for 1h. Amiloride was added 30 minutes before to block macropinocytosis. Percentage of macropinocytic cells was measured by flow cytometry. (E) Cells were fixated, permeabilized and stained with anti-CD14 Alexa Fluor 647 (macrophages) and anti-CD61 PE (platelets) antibodies. DAPI (nuclei) was employed as counterstain and confocal images were obtained. The white boxes correspond to the magnified images on the right (orthogonal views showing platelets (CD61) and internalized CD H FITC in macrophages (DIC/DAPI)). In the panoramic micrographs, white arrowheads point macrophages internalizing CD H. DAPI (nuclei) is shown in blue, CD61 (platelets) in red and CD14 (macrophages) in purple. DIC: differential interference contrast. Scale bar: 10 μm. (F–H) MΦs were stimulated with CD H FITC or CD H FITC plus NAP1/BI/027 secretome ( CD H FITC+Sec) or infected with live CD160 FITC (non-toxigenic) or NAP1/BI/027 FITC (hypervirulent) C. difficile strains as stated in (D) . (A–C, E) correspond to three individual donors in three independent experiments, (D) correspond to nine individual donors in 9 independent experiments. (F–H) correspond to four donors in independent experiments. (A) Violin plots, (D, F–H) Bars represent the mean ± SEM. (D) Each dot corresponds to an individual healthy donor. (A, D) Friedman test. (F–H) 2-way ANNOVA *p<0.05, **p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: In vitro infection of cells was performed with vegetative live non-toxigenic CD160 (NR-43516, BEI Resources) or hypervirulent NAP1/BI/027 (Sanitary Bacteriology Service INEI-ANLIS, Dr. Carlos G. Malbrán, Argentina) C. difficile strains.

Techniques: Cell Culture, Flow Cytometry, Confocal Microscopy, Staining, Blocking Assay, Infection