Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: Cytotoxic necrotizing factor 1 (CNF1), but not CNF1-C866S, induces phenotypic maturation of immature monocyte-derived dendritic cells (moDCs). ( A – C ) Immature moDCs were cultured for 24 and 48 h without toxin (Ctrl) or with different concentrations of CNF1 or CNF1-C866S before the expression of phenotypic maturation markers was measured by flow cytometry. ( A ) Representative pseudocolor dot plot showing surface expression of CD83 and CD86. ( B ) Graphs summarizing the mean percentages of CD83+CD86+ cells from 3 independent experiments, each using moDCs derived from different donors ( n = 3). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to immature moDCs cultured without toxin using a two-way ANOVA with Sidak’s multiple comparisons test. ( C ) Histogram plots showing the surface expression of HLA-DR on moDCs cultured without toxin (green), with CNF1 (red) or with CNF1-C866S (blue). Arrows indicate increasing concentrations (0.004, 0.02 and 0.1 nM) of CNF1 and CNF1-C866S. The plots are representative of 3 independent experiments each using moDCs derived from different donors ( n = 3).

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Derivative Assay, Cell Culture, Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1-induced moDC maturation is not driven by endotoxin or other putative contaminants in the toxin preparation. ( A ) Culture media alone (Ctrl) or culture media containing 0.1 nM CNF1, 50 ng/mL lipopolysaccharide (LPS) or 50 ng/mL tumor necrosis factor alpha (TNFα) was pre-incubated for 30 min at room temperature with 5 µg/mL Polymyxin B (PMB) or vehicle (endotoxin-free water). Then, immature moDCs were added to each media sample and cultured for 24 h before the expression of CD83 and CD86 was determined by flow cytometry. Shown is the mean percentage of CD83+CD86+ moDCs from 4 different donors ( n = 4) analyzed in 3 independent experiments. Error bars represent SEM and * denotes a significant difference ( p < 0.05) between vehicle- and PMB-treated cells using a paired Student’s t -test. ( B ) CNF1 diluted in moDC medium was incubated with (2) uncoated Protein A agarose beads or (3) Protein A agarose beads that had been pre-coated with a monoclonal anti-CNF1 (αCNF1) antibody or (4) an isotype control (IC) antibody. As a negative control, (1) moDC medium without CNF1 was in parallel incubated with uncoated Protein A agarose beads. After removal of the beads, the level of CNF1 in the supernatants was analyzed by western blotting. ( C ) Immature moDCs were cultured for 24 h with the supernatants (1–4) obtained in ( B ) and the expression of CD83 and CD86 analyzed by flow cytometry.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Incubation, Cell Culture, Expressing, Flow Cytometry, Negative Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1 does not induce apoptosis or cell death in moDCs. ( A , B ) Immature moDCs were cultured for 24 and 48 h without toxin (Ctrl) or with 0.1 nM CNF1 before the percentages of Annexin V- and propidium iodide (PI)-positive cells were determined by flow cytometry. moDCs cultured in medium without fetal bovine serum and cytokines (Starved) were included in the analysis as a positive control for induction of apoptotic cell death. ( A ) Histogram summarizing the mean percentages of Annexin V+ and/or PI+ cells from 3 independent experiments, each using moDCs derived from different donors ( n = 3). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to Ctrl moDCs at the same timepoint using a two-tailed paired Student’s t -test. ( B ) A representative pseudocolor dot plot of an Annexin V and PI staining after 48 h of culture.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Cell Culture, Flow Cytometry, Positive Control, Derivative Assay, Two Tailed Test, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1 induces functional maturation of moDCs. ( A , B ) Immature moDCs were cultured for 24 h without toxin (Ctrl), with 0.1 nM CNF1 or with 0.1 nM CNF1-C866S before the concentrations of ( A ) interleukin-6 (IL-6) and ( B ) TNFα in the cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). ( A , B ) Shown is the mean cytokine concentration + SEM in cell culture supernatants from 3 independent experiments each using moDCs derived from different donors ( n = 3). ( C ) Immature moDCs were cultured for 48 h with 0.1 nM CNF1, 0.1 nM CNF1-C866S, 50 ng/mL LPS or without toxin (Ctrl). The moDCs were subsequently washed and different numbers cultured with allogenic naïve CD4+ T cells for 4 days. Then [ 3 H]-thymidine was added, and the cells cultured for 24 h further, before determination of [ 3 H]-thymidine incorporation. Shown are the mean counts per minute (CPM) from 3 independent experiments. Each independent experiment was performed using moDCs and allogenic naïve CD4+ T cells derived from different donors (total of 6 donors). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to CD4+ T cells cultured with the same number of Ctrl moDCs using a two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Functional Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: Design, expression, and solubility of cytotoxic necrotizing factor 1 (CNF1) variants. ( A ) Scheme of engineered CNF1 constructs presenting the following N-terminal and C-terminal tags: Angiopep-2 (An2), spaced by the GGSSRSS linker; 8xHis fused to a Tobacco Etch Virus recognition site (H8). ( B ) Recombinant protein expression levels in E. coli BL21 (DE3). Total cellular extracts obtained at 15 °C after 18 h induction with 0.5 mM IPTG were separated by 10% SDS-PAGE and analyzed either by Coomassie staining ( B1 ) or western blot ( B2 ) using a monoclonal anti-CNF1 antibody (NG8). Induced cells expressing CNF1-H8; induced cells expressing An2-CNF1-H8; not induced recombinant cells harboring pET40b- cnf1-h8 . ( C ) Evaluation of CNF1 solubility in E. coli extracts. After cellular lysis, insoluble (P) and soluble (S) fractions were analyzed. The first two panels are relative to the solubility assay concerning CNF1-H8 expression carried out using SDS-PAGE ( C1 and C3 ) and western blot ( C2 and C4 ). The third and fourth images represent the SDS-PAGE and western blot relative to An2-CNF1-H8 solubility, respectively.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Expressing, Solubility, Construct, Virus, Recombinant, SDS Page, Staining, Western Blot, Lysis

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: CNF1 variants activity. ( A ) SDS-polyacrylamide gel ( A1 ) and western blot ( A2 ) of CNF1 variants. ( B ) Phase-contrast micrographs of HEp-2 cells treated with CNF1 variants, showing the multinucleating/ruffling effects. Scale bar: 10 μm.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: Entry of CNF1 variants into HEp-2 cells. ( A ) Graph showing the inhibition of variants entry by mCNF1. ( B ) Western blot analysis of HEp-2 cells treated with An2-CNF1-H8 and its controls (CNF1-H8 and wt CNF1) and stained with an antibody against CNF1, normalized as a function of GAPDH. Note that all the toxins are found as full length and 55 kDa C-ter portions after 24 h treatment ( B1 ). Over a longer period of time, that is 48 h of treatment, washing to eliminate any remaining molecules in the culture medium, and further culture for 24 h, the full-length portions completely disappeared while the 55 kDa C-ter portions were present at a much lower concentration ( B2 ). All in all, these results point at the fact that An2-CNF1-H8 follows the same route of entry of wt CNF1. Variants and wt CNF1 were used at 10 −10 M. Data on the graph represent the mean ± SEM from at least three independent experiments. *** p < 0.001.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Inhibition, Western Blot, Staining, Concentration Assay

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: An2-CNF1-H8 activity on endothelial cells. Fluorescence micrographs of HBEC-5i cells treated with the An2-CNF1-H8 variant and its controls. Note the same actin architecture in cells exposed to the wt CNF1 and to the variants, indicating the same activity. Scale bar: 10 μm.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Activity Assay, Fluorescence, Variant Assay

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: An2-CNF1-H8 impairs growth in U87MG GBM cells. ( A and B ) Graphs showing cell growth impairment by An2-CNF1-H8 ( A ) and CNF1 ( B ) on U87MG GBM cells. Note that, whereas controls cells increase in cell number in the function of time, cells treated with An2-CNF1-H8 and CNF1 stopped growing. Data on the graph represent the mean ± SEM from at least three independent experiments. *** p < 0.001.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques:

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: Mitochondrial impact and toxicity of An2-CNF1-H8 on U87MG cells. ( A ) Fluorescence micrographs of U87MG cells treated with An2-CNF1-H8 and stained with phalloidin, to detect F-actin, Mitotracker, to detect mitochondria and Hoechst 33342 to stain nuclei. Note that the variant impacts on mitochondria that appear slightly fragmented and scattered throughout the cell body with respect to controls. ( B and C ) Western blot of U87MG cells treated with An2-CNF1-H8 and graph analyses, showing that ( B1 and B2 ) Tom20 was transiently increased, whereas OPA-1 protein was not modified by the An2-CNF1-H8; ( C1 and C2 ) the pro-apoptotic Bax protein, involved in mitochondrial outer membrane permeabilization, was increased whereas the senescence marker beta-gal remained unvaried. Proteins were normalized as a function of GAPDH. ** p < 0.01. Scale bar: 10 μm.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Fluorescence, Staining, Variant Assay, Western Blot, Modification, Membrane, Marker