Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Internalization of fluorescein isothiocyanate-conjugated monoclonal anti-human CCR7 induced by recombinant CCL19 in intact HEK 293a cells that express recombinant CCR7. nt, non-transfected cells.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Transfection

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Endocytosis of the anti-myc monoclonal antibody (clone 4A6, conjugated to AlexaFluor-488, final concentration in the culture medium 3.3 nM) as determined by co-treatment with the CCL19-myc construction in intact HEK 293a cells that optionally and transiently expressed CCR7. A control conditioned medium (CM) or authentic CCL19 were used as control stimuli. The undiluted CM were transferred for a 30-min incubation period before rinsing and observation. Original magnification ×1000.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Concentration Assay, Control, Incubation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Cytofluorometry of HEK 293a cells that optionally expressed CCR7, sequentially detached and further stimulated with the undiluted CM of other cells producing CCL19-myc, authentic CCL19, or CM of untransfected cells, as indicated (30 min incubation at 37°C). Left: distributions based on the counting of 10,000 cells. A threshold of autofluorescence was defined using control cells with no fluorophore. It was surpassed only under one set of experimental conditions (arrow). Right: mean fluorescence of cells in replicated experiment. ANOVA indicated that the values were heterogeneous ( p < 10 -4 ). * p < 0.01 vs. top-most value (control CM, untransfected recipient cells) by Dunnett’s test.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Control, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing β-arrestin 1 -Cherry and CCR7 and stimulated as indicated (stimulant and duration). Stimulation always included the fluorescent anti-myc monoclonal antibody 4A6 (green signal). Either CCL19-myc CM or authentic CCL19 rarely condensed β-arrestin 1 at the level of plasma membranes of endosomes (arrowhead indicates possible co-localization with the CCL19-myc-antibody cargo). Cells that expressed well the red-emitting transgene were assumed to co-express co-transfected CCR7 and were evaluated for morphology ( n = 16–49 eligible cells per group). The average number of red condensed structures was 0.4 ± 0.4 in cells treated with control CM and did not vary significantly in all other groups (Kruskal–Wallis test). The average number of green condensed structures in cells treated with control CM was 0 per cell and varied significantly according to treatments ( p < 10 -4 , Kruskal–Wallis test; p values for Dunn’s multiple comparison test for each value vs. that of control CM reported thereafter). There were 8.3 ± 1.7 green specs per cell ( p < 0.05), 25.8 ± 3.2 ( p < 0.001), 33.1 ± 4.4 ( p < 0.001), and 0 (N.S.) in cells treated with CCL19-myc CM for 5, 10, or 30 min, or with recombinant CCL19, respectively.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Clinical Proteomics, Transfection, Control, Comparison, Recombinant

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing CCR7 and β-arrestin 2 -GFP and stimulated as indicated (stimulant and duration). Stimulation did not include the fluorescent anti-myc antibody. Either CCL19-myc CM or authentic CCL19 frequently condensed β-arrestin 2 at the level of plasma membrane or endosomes. Right: number of green intracellular condensed structures (“specs”) per cell ± SEM. Numbers close to bars indicate the number of evaluated HEK 293a cells. The Kruskal–Wallis test indicated that the values were heterogenous ( p < 10 -4 ). The effect of each treatment vs. the effect of control CM was evaluated using Dunn’s multiple comparison test. * p < 0.01; ** p < 0.001.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Clinical Proteomics, Membrane, Control, Comparison

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing one of three forms of Rab5-Cherry fusion proteins and CCR7 and stimulated for 30 min with either CCL19-myc or control CM. Co-localization between Rab5-positive corpuscles and the CCL19-myc-antibody cargo was observed (arrowheads). The dominant positive (GTP-locked) mutant of the fusion protein produced typical giant vacuoles that included the green light-emitting cargo. The dominant negative Rab5-GDP-locked-Cherry inhibited the endocytosis of the green cargo (see text for numerical analysis).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Control, Mutagenesis, Produced, Dominant Negative Mutation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing Rab7-Cherry and CCR7 and stimulated as indicated (stimulant and duration). Co-localization between Rab7-positive corpuscles and the CCL19-myc-antibody cargo was not highly frequent (arrowheads), but increased in frequency as a function of incubation duration (see text for numerical analysis).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Incubation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Presence of CCR7 in A375 melanoma cells. (A) RT-PCR for CCR7 in the human melanoma A375 cell line. (B) c-Fos induction in A375 cells stimulated as indicated for 1 h (typical immunoblot and histograms representing the densitometry of replicated experiments).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Detection of endogenous CCR7 in A375 cells using detection of the endocytosed CCL19-myc-4A6 antibody complex using theTyramide Signal Amplification (TSA) system that enzymatically generates AlexaFluor-488 labeling. Epifluorescence and transmission, original magnification: ×1000. Right: proportion of cells with a mean fluorescence intensity above a set threshold (Photoshop level ≥ 50/255) in large photographic records of A375 cells stained as in the left of figure (the number of evaluated cells is indicated next to each histogram). The proportions were compared with that of control cells without antibody (top-most histogram, χ 2 test; * p < 10 -4 ).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Amplification, Labeling, Transmission Assay, Fluorescence, Staining, Control

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 3. Doxorubicin-induced apoptosis in leukemic cells is dependent on Rac1. (A,B) The Rac1 inhibitor reduces doxorubicin-induced apoptosis in U937 and Jurkat cells. The cells were pretreated with the specific Rac1 inhibitor (NSC23766, 10 μM, 1 h), before their treatment with doxorubicin (Dox) for 24 h. Apoptosis was determined by annexin V staining and flow cytometry analysis. (C,D) Overexpression of the Rac1 dominant negative form N17Rac1 inhibits doxorubicin-induced apoptosis. The cells were transfected with pcDNA3.1, N17Rac1 + pcDNA3.1 or with N17Rac1 + WT-Rac1 plasmids. Viable cells were recovered after 24 h by ficoll gradient. The cells were then treated with doxorubicin for 24 h. Apoptosis was determined by annexin V staining and flow cytometry analysis. The results represent mean values ± SD from three independent experiments. *P < 0.05, **P < 0.01. (E) Rac1 inhibition blocks caspase-9 and -3 activation by doxorubicin. Jurkat cells were treated as indicated and after 12 h of doxorubicin treatment, the levels of native and active caspase-9 and -3 were determined by western blot analysis. β-actin was used as a loading control. The illustrated blots are representative of three independent experiments.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Staining, Flow Cytometry, Over Expression, Dominant Negative Mutation, Transfection, Inhibition, Activation Assay, Western Blot, Control

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 4. Collagen inhibits doxorubicin-induced Rac1 activation via α2β1 integrin. U937 (A) and Jurkat (B) cells were cultured on BSA (-) or on collagen (Col) and then treated or not with doxorubicin (Dox) for 3 h. The cells were harvested, lysed and Rac1 activation was determined by Rac1-GLISA assay. (C) The effect of collagen is mediated via α2β1 integrin. Jurkat cells were treated as above except that before their culture on collagen they were pretreated with 10 μg/ml of control IgG or with the blocking anti-α2 integrin mAb (P1E6). The results represent mean values ± SD from three independent experiments. **P < 0.01.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Activation Assay, Cell Culture, Control, Blocking Assay

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 5. Collagen inhibits Rac1 activation independently from ABCC1. (A) ABCC1 protein levels in Jurkat T cells transfected with control and specific ABCC1 siRNA levels. The immunoblot is representative of three independent experiments. (B) ABCC1 silencing does not affect collagen-mediated Rac1 inhibition. Control and ABCC1 siRNA-transfected Jurkat T cells were cultured on BSA or on collagen and then treated with doxorubicin (Dox) for 3 h. The cells were harvested, lysed and Rac1 activation was determined by Rac1-GLISA assay. (C) The ABCC1 inhibitor MK571 does not affect collagen-mediated Rac1 inhibition in U937 cells. The cells were pretreated with the vehicle or MK571 (10 μM) for 1 h after which, the cells were cultured on BSA or collagen and then treated with Dox. Rac1 activation was determined by Rac1-GLISA assay. The results represent mean values ± SD from three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Activation Assay, Transfection, Control, Western Blot, Inhibition, Cell Culture

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 6. Rac1 inhibition reduces DNA damage intensity and H2AX phosphorylation induced by doxorubicin. (A–C) The cells were treated or not with doxorubicin (Dox) for 6 h in the presence or absence of the Rac1 inhibitor NSC23766 (NSC). Alkaline comet assay was performed and stained nucleoids were visualized by epifluorescence microscopy using FITC filter. (A) Representative fields corresponding to each treatment were photographed. (B,C) The intensity of DNA strand breaks in U937 and Jurkat cells was quantified using visual scoring as described under “Experimental procedures section”. The results represent mean values ± SD obtained from three independent experiments. **P < 0.01. (D,E) The cells were treated with doxorubicin in the presence or absence of NSC23766 as described above and the levels of phosphorylated H2AX (γ-H2AX) were determined by immunoblot analysis. The β-Actin blot was used as a loading control. Blots are representative of three independent experiments.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Inhibition, Phospho-proteomics, Alkaline Single Cell Gel Electrophoresis, Staining, Epifluorescence Microscopy, Western Blot, Control

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 8. Rac1 is involved in doxorubicin-induced JNK activation and Mcl-1 downregulation. U937 and Jurkat cells were treated or not with doxorubicin (Dox) in the presence or absence of the Rac1 inhibitor NSC23766. After 8 h of treatment, the cells were lysed and the levels of phospho-JNK1/2 (A&B) and Mcl-1 (C&D) were determined by immunoblot analysis. The blots were stripped and reprobed with anti-β-actin antibody for equal loading. The blots are representative of three independent experiments.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Development of intimin-enriched outer membrane vesicles (OMVs) as a vaccine to control intestinal carriage of Enterohemorrhagic Escherichia coli

doi: 10.1101/2024.12.09.627534

Figure Lengend Snippet: Addressing of C. rodentium Int280 to the lumen and surface of OMVs using PelB- and Lpp-OmpA-based systems. ( A ) Schematic representation of the recombinant plasmids and corresponding OMVs containing Int280 in their lumen or on their surface. The pET-22b(+) expression vector was used to express the PelB-Int280 (P-Int280) or Lpp-OmpA-Int280 (LO-Int280) fusion proteins under control of the T7 promoter. The PelB signal peptide addresses Int280 in the lumen of OMVs (left) while the hybrid protein Lpp-OmpA addresses Int280 to the surface of OMVs (right). The amino acids (AA) of each protein fragment incorporated in the fusion proteins are indicated. ( B ) Detection of P-Int280 and LO-Int280 in OMVs fractions. OMVs were obtained from E. coli BL21(DE3) Δ ompF containing plasmid pET-22b(+) (“Empty vector”), pAGA12 (“P-Int280”) or pAGA16 (“LO-Int280”). Bacterial cultures were induced with 1 mM of IPTG. An equivalent amount of OMVs (as determined by total protein assay) was loaded in each lane for analysis by SDS-PAGE stained with Coomassie Blue (10 µg OMVs extracts) (left panel) and Western blotting (2 µg OMVs extracts) (right panel). For Western blotting, membrane was probed with an anti-Int280 antibody. Locations of P-Int280 (30kDa) and LO-Int280 (43kDa) are indicated with arrowheads.

Article Snippet: The resulting recombinant plasmids were introduced into NEB 5-alpha competent E. coli cells by thermal shock and transformed strains were incubated at 37°C for 1h.

Techniques: Recombinant, Expressing, Plasmid Preparation, Control, SDS Page, Staining, Western Blot, Membrane

Journal: bioRxiv

Article Title: Development of intimin-enriched outer membrane vesicles (OMVs) as a vaccine to control intestinal carriage of Enterohemorrhagic Escherichia coli

doi: 10.1101/2024.12.09.627534

Figure Lengend Snippet: Int280 fused to Lpp-OmpA protein is addressed to the bacterial surface and restores the actin polymerization phenotype of the EPEC E22 intimin-mutant strain. Actin polymerization induced by the EPEC E22 strain in HeLa cells was observed with the Fluorescent Actin Staining (FAS) assay. HeLa cells were infected 5h with EPEC E22 WT, EPEC E22 Δ eae + pBRSK or EPEC E22 Δ eae + pBRSK_LO-Int280 (pAGA43) strains at a MOI of 50 bacteria per cell. Following staining of F-actin with rhodamine-phalloidin and bacteria with anti- E. coli antibodies, images were captured by epifluorescence microscopy. The lower panels show enlarged microscopy fields. Actin polymerization in some pedestals (red) beneath the bacteria (green) is indicated by arrowheads. Images are representative of three independent experiments.

Article Snippet: The resulting recombinant plasmids were introduced into NEB 5-alpha competent E. coli cells by thermal shock and transformed strains were incubated at 37°C for 1h.

Techniques: Mutagenesis, Staining, Infection, Bacteria, Epifluorescence Microscopy, Microscopy

Journal: bioRxiv

Article Title: Development of intimin-enriched outer membrane vesicles (OMVs) as a vaccine to control intestinal carriage of Enterohemorrhagic Escherichia coli

doi: 10.1101/2024.12.09.627534

Figure Lengend Snippet: The OMV-Int280 vaccine elicited an anti-intimin IgG immune response in mice. ( A ) Experimental timeline of immunization experiment with female C57BL/6 mice. One group of 5 mice received PBS as a control and one group of 5 mice received the OMV-P-Int280/OMV-LO-Int280 mixed vaccine (OMV-Int280). The syringes indicate the days of immunization. Blood samples were collected on the indicated days for serum preparation. ( B ) Seroconversion of mice observed by Western Blotting with bacterial lysates of C. rodentium ICC792 (Δ eae ) or C. rodentium ICC169, or with OMV-P-Int280 (2 µg) or OMV-LO-Int280 (2 µg) extracts obtained from E. coli BL21(DE3) Δ ompF carrying plasmid pAGA12 or pAGA16, respectively. “Empty vector” corresponds to the OMVs extracts obtained from E. coli BL21(DE3) Δ ompF carrying the empty pET-22b(+) plasmid. Sera collected from either PBS or vaccinated mice groups were each pooled and used to detect (i) full-length intimin (94kDa) from C. rodentium ICC169 bacterial lysates (top), or (ii) P-Int280 (30kDa) and LO-Int280 (43kDa) from OMVs fractions (bottom). The locations of intimin, P-Int280 and LO-Int280 are indicated with arrowheads. Anti-Int280 antibody was used as a positive control (top and bottom, third blot).

Article Snippet: The resulting recombinant plasmids were introduced into NEB 5-alpha competent E. coli cells by thermal shock and transformed strains were incubated at 37°C for 1h.

Techniques: Control, Western Blot, Plasmid Preparation, Positive Control