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    ATCC vk2 e6e7 cells
    Subcellular location of intracellular bacteria in mitosis. A. <t>VK2/E6E7</t> cells were infected with DyLight 594 NHS Ester-stained N. gonorrhoeae . Cells were maintained in a live-cell incubator connected to an inverted fluorescence microscope. Cellular DNA was stained with Hoechst 33342 and is shown in blue. Images of live mitotic cells were taken through a 100x oil objective in 40–60 z-stacks. Images were further deconvolved and processed by Inside 4D software. Bacteria (red) present during prophase (upper), prometaphase (mid) and anaphase (lower) are shown. Bacteria that are in close proximity to the chromatin are marked with arrows. The scale bar represents 10 µm. B. Viable bacteria associate with chromatin in ELISA. Chromatin, extracted from 1.25×10 5 cells was coated in each well. Viable (5×10 6 CFU/well or 1×10 6 CFU/well) or PFA-fixed (5×10 6 CFU/well) MS11P+ was allowed to interact with the chromatin for 2 h at 37°C and 5% CO 2 . Rabbit anti- N. gonorrhoeae antibodies followed by goat anti rabbit IgG-HRP antibodies were used to detect the bacteria bound to the chromatin. Absorbance was read at 450 nm. Background was measured in wells containing all agents except chromatin. Shown is a graph of one assay in technical triplicate and standard deviations.
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    Subcellular location of intracellular bacteria in mitosis. A. VK2/E6E7 cells were infected with DyLight 594 NHS Ester-stained N. gonorrhoeae . Cells were maintained in a live-cell incubator connected to an inverted fluorescence microscope. Cellular DNA was stained with Hoechst 33342 and is shown in blue. Images of live mitotic cells were taken through a 100x oil objective in 40–60 z-stacks. Images were further deconvolved and processed by Inside 4D software. Bacteria (red) present during prophase (upper), prometaphase (mid) and anaphase (lower) are shown. Bacteria that are in close proximity to the chromatin are marked with arrows. The scale bar represents 10 µm. B. Viable bacteria associate with chromatin in ELISA. Chromatin, extracted from 1.25×10 5 cells was coated in each well. Viable (5×10 6 CFU/well or 1×10 6 CFU/well) or PFA-fixed (5×10 6 CFU/well) MS11P+ was allowed to interact with the chromatin for 2 h at 37°C and 5% CO 2 . Rabbit anti- N. gonorrhoeae antibodies followed by goat anti rabbit IgG-HRP antibodies were used to detect the bacteria bound to the chromatin. Absorbance was read at 450 nm. Background was measured in wells containing all agents except chromatin. Shown is a graph of one assay in technical triplicate and standard deviations.

    Journal: PLoS ONE

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    doi: 10.1371/journal.pone.0114208

    Figure Lengend Snippet: Subcellular location of intracellular bacteria in mitosis. A. VK2/E6E7 cells were infected with DyLight 594 NHS Ester-stained N. gonorrhoeae . Cells were maintained in a live-cell incubator connected to an inverted fluorescence microscope. Cellular DNA was stained with Hoechst 33342 and is shown in blue. Images of live mitotic cells were taken through a 100x oil objective in 40–60 z-stacks. Images were further deconvolved and processed by Inside 4D software. Bacteria (red) present during prophase (upper), prometaphase (mid) and anaphase (lower) are shown. Bacteria that are in close proximity to the chromatin are marked with arrows. The scale bar represents 10 µm. B. Viable bacteria associate with chromatin in ELISA. Chromatin, extracted from 1.25×10 5 cells was coated in each well. Viable (5×10 6 CFU/well or 1×10 6 CFU/well) or PFA-fixed (5×10 6 CFU/well) MS11P+ was allowed to interact with the chromatin for 2 h at 37°C and 5% CO 2 . Rabbit anti- N. gonorrhoeae antibodies followed by goat anti rabbit IgG-HRP antibodies were used to detect the bacteria bound to the chromatin. Absorbance was read at 450 nm. Background was measured in wells containing all agents except chromatin. Shown is a graph of one assay in technical triplicate and standard deviations.

    Article Snippet: Cell lines and growth conditions The immortalized human vaginal epithelial cell line VK2/E6E7 (ATCC CRL-2616, LGC Standards, London) has been derived from normal vaginal mucosal tissue and shows characteristics of stratified squamous non-keratinizing epithelia.

    Techniques: Infection, Staining, Fluorescence, Microscopy, Software, Enzyme-linked Immunosorbent Assay

    Gonococcal infection causes durations of mitosis, nuclear swelling, and targets regulatory mitotic genes and proteins. A. VK2/E6E7 cells were synchronized and bacteria were allowed to adhere and invade cells for 24 h. Cells were then fixed and stained with DAPI. The nuclear area of 300 infected and 300 uninfected cells were measured. Data were analyzed using a paired 2-tailed Student's t -test. The average nuclear areas observed in 3 independent experiments are shown (* p

    Journal: PLoS ONE

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    doi: 10.1371/journal.pone.0114208

    Figure Lengend Snippet: Gonococcal infection causes durations of mitosis, nuclear swelling, and targets regulatory mitotic genes and proteins. A. VK2/E6E7 cells were synchronized and bacteria were allowed to adhere and invade cells for 24 h. Cells were then fixed and stained with DAPI. The nuclear area of 300 infected and 300 uninfected cells were measured. Data were analyzed using a paired 2-tailed Student's t -test. The average nuclear areas observed in 3 independent experiments are shown (* p

    Article Snippet: Cell lines and growth conditions The immortalized human vaginal epithelial cell line VK2/E6E7 (ATCC CRL-2616, LGC Standards, London) has been derived from normal vaginal mucosal tissue and shows characteristics of stratified squamous non-keratinizing epithelia.

    Techniques: Infection, Staining

    Bacterial infection induces micronuclei formation in VK2/E6E7 cells. VK2/E6E7 cells were infected with N. gonorrhoeae for 24 h. Cytokinesis was blocked with cytochalasin B for 36 h and BNC from infected and control cells were analyzed for micronuclei formation. A. Average numbers of observed micronuclei/1000 BNC ± standard deviation from 3 independent experiments are shown (* p

    Journal: PLoS ONE

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    doi: 10.1371/journal.pone.0114208

    Figure Lengend Snippet: Bacterial infection induces micronuclei formation in VK2/E6E7 cells. VK2/E6E7 cells were infected with N. gonorrhoeae for 24 h. Cytokinesis was blocked with cytochalasin B for 36 h and BNC from infected and control cells were analyzed for micronuclei formation. A. Average numbers of observed micronuclei/1000 BNC ± standard deviation from 3 independent experiments are shown (* p

    Article Snippet: Cell lines and growth conditions The immortalized human vaginal epithelial cell line VK2/E6E7 (ATCC CRL-2616, LGC Standards, London) has been derived from normal vaginal mucosal tissue and shows characteristics of stratified squamous non-keratinizing epithelia.

    Techniques: Infection, Standard Deviation

    Microinjection of bacterial lysates in the cytoplasm of VK2/E6E7 cells causes DSBs. A. Interphase VK2/E6E7 cells were subjected to cytoplasmic microinjection of bacterial MS11 P+ lysate, MS11 P+ HI lysate, or PBS. Cells were incubated for 20–24 h and then stained for DSBs with 53BP1 antibodies. The graph shows the average number of 53BP1 positive cells counted in two independent experiments under each condition. Control cells are non-injected cells. B. Images showing DIC and fluorescent images of representative cells microinjected with MS11 P+ lysate (left) or PBS (right). FITC-dextran (green) was co-injected into the cytoplasm to identify microinjected cells. Scale bar represents 10 µm.

    Journal: PLoS ONE

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    doi: 10.1371/journal.pone.0114208

    Figure Lengend Snippet: Microinjection of bacterial lysates in the cytoplasm of VK2/E6E7 cells causes DSBs. A. Interphase VK2/E6E7 cells were subjected to cytoplasmic microinjection of bacterial MS11 P+ lysate, MS11 P+ HI lysate, or PBS. Cells were incubated for 20–24 h and then stained for DSBs with 53BP1 antibodies. The graph shows the average number of 53BP1 positive cells counted in two independent experiments under each condition. Control cells are non-injected cells. B. Images showing DIC and fluorescent images of representative cells microinjected with MS11 P+ lysate (left) or PBS (right). FITC-dextran (green) was co-injected into the cytoplasm to identify microinjected cells. Scale bar represents 10 µm.

    Article Snippet: Cell lines and growth conditions The immortalized human vaginal epithelial cell line VK2/E6E7 (ATCC CRL-2616, LGC Standards, London) has been derived from normal vaginal mucosal tissue and shows characteristics of stratified squamous non-keratinizing epithelia.

    Techniques: Incubation, Staining, Injection

    Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

    Journal: PLoS ONE

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    doi: 10.1371/journal.pone.0114208

    Figure Lengend Snippet: Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

    Article Snippet: Cell lines and growth conditions The immortalized human vaginal epithelial cell line VK2/E6E7 (ATCC CRL-2616, LGC Standards, London) has been derived from normal vaginal mucosal tissue and shows characteristics of stratified squamous non-keratinizing epithelia.

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Purification, Negative Control, Incubation

    Spores of the csB mutant show increased adherence to cervical but not vaginal cells compared to wild-type spores. Cervical cell lines Ect1/E6E7 (A) and End1/E6E7 (B) and vaginal cell line Vk2/E6E7 (C) were incubated with spores for 3 hours, washed to remove unbound spores and the number of bound spores determined. The results represent the average of four independent experiments and error bars represent standard error of the mean. Asterisks indicate statistical difference at p ≤ 0.05. Wild-type, WT; csA mutant, csA -M; csB mutant, csB -M; csB mutant complemented, csB -C.

    Journal: PLoS Pathogens

    Article Title: Clostridium sordellii outer spore proteins maintain spore structural integrity and promote bacterial clearance from the gastrointestinal tract

    doi: 10.1371/journal.ppat.1007004

    Figure Lengend Snippet: Spores of the csB mutant show increased adherence to cervical but not vaginal cells compared to wild-type spores. Cervical cell lines Ect1/E6E7 (A) and End1/E6E7 (B) and vaginal cell line Vk2/E6E7 (C) were incubated with spores for 3 hours, washed to remove unbound spores and the number of bound spores determined. The results represent the average of four independent experiments and error bars represent standard error of the mean. Asterisks indicate statistical difference at p ≤ 0.05. Wild-type, WT; csA mutant, csA -M; csB mutant, csB -M; csB mutant complemented, csB -C.

    Article Snippet: Spores were tested for their ability to adhere to or be internalized by the following cell lines: VK2/E6E7 vaginal epithelial cell line (ATCC CRL-2616), End1/E6E7 endocervical epithelial cell line (ATCC CRL-2615) and Ect1/E6E7 ectocervical epithelial cell line (ATCC CRL-2614).

    Techniques: Mutagenesis, Incubation

    Effect of bacterium–host cell interaction on the tolerance of human vaginal epithelial cells to antimicrobial blue light (aBL) and the susceptibility of Neisseria gonorrhoeae to aBL inactivation. A , Confocal image of vaginal epithelial cells (VK2/E6E7) infected with N. gonorrhoeae (ATCC 700825). Both VK2/E6E7 cells and N. gonorrhoeae were stained green by SYTO9, and N. gonorrhoeae cells are depicted as green dots (arrows). N. gonorrhoeae cells either adhered to the cell walls or invaded the cytoplasm of the VK2/E6E7 cells. B , Viability changes among human vaginal epithelial cells (VK2/E6E7) infected by N. gonorrhoeae in response to aBL . C , Viability changes among intracellular N. gonorrhoeae in response to aBL.

    Journal: The Journal of Infectious Diseases

    Article Title: Photoinactivation of Neisseria gonorrhoeae: A Paradigm-Changing Approach for Combating Antibiotic-Resistant Gonococcal Infection

    doi: 10.1093/infdis/jiz018

    Figure Lengend Snippet: Effect of bacterium–host cell interaction on the tolerance of human vaginal epithelial cells to antimicrobial blue light (aBL) and the susceptibility of Neisseria gonorrhoeae to aBL inactivation. A , Confocal image of vaginal epithelial cells (VK2/E6E7) infected with N. gonorrhoeae (ATCC 700825). Both VK2/E6E7 cells and N. gonorrhoeae were stained green by SYTO9, and N. gonorrhoeae cells are depicted as green dots (arrows). N. gonorrhoeae cells either adhered to the cell walls or invaded the cytoplasm of the VK2/E6E7 cells. B , Viability changes among human vaginal epithelial cells (VK2/E6E7) infected by N. gonorrhoeae in response to aBL . C , Viability changes among intracellular N. gonorrhoeae in response to aBL.

    Article Snippet: Human Vaginal Epithelial Cells and Growth Conditions Human vaginal epithelial cells VK2/E6E7 (ATCC CRL-2616) were used.

    Techniques: Infection, Staining

    POD-NLC inhibits cell proliferation in VK2/E6E7 cells. (A) Comparison of inhibitory effects of different concentrations of POD-NLC and free-POD following 48 h of treatment. Ns, no significant difference vs. POD group; *P

    Journal: Molecular Medicine Reports

    Article Title: Development of podophyllotoxin-loaded nanostructured lipid carriers for the treatment of condyloma acuminatum

    doi: 10.3892/mmr.2018.8696

    Figure Lengend Snippet: POD-NLC inhibits cell proliferation in VK2/E6E7 cells. (A) Comparison of inhibitory effects of different concentrations of POD-NLC and free-POD following 48 h of treatment. Ns, no significant difference vs. POD group; *P

    Article Snippet: The immortalized vaginal epithelial cells VK2/E6E7 (ATCC® CRL-2616™) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in an atmosphere containing 5% CO2 .

    Techniques:

    Intracellular production of C. trachomatis RBs in VK2/E6E7 cells. Cells were incubated with PBS, non-silencing siRNA PLGA-PEG NP (1.334 mg/mL), non-silencing siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) or PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL) and autophagy inhibitor (bafilomycin A, 50 mM), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) and autophagy inducer (rapamycin, 100 nM) for 48 hr and then infected with C. trachomatis. Images were taken 24 hr post C. trachomatis infection. ( A ) Intracellular C. trachomatis RB foci (green fluorescence) were visualized using fluorescence microscopy. Experiments were conducted n = 3 and a group of representative images were shown. ( B ) Semi-quantitative measurements of intracellular C. trachomatis RB foci were accomplished using Image J software. Values represent the mean ± SD, n = 3. *p

    Journal: Scientific Reports

    Article Title: Autophagy induction and PDGFR-β knockdown by siRNA-encapsulated nanoparticles reduce chlamydia trachomatis infection

    doi: 10.1038/s41598-018-36601-y

    Figure Lengend Snippet: Intracellular production of C. trachomatis RBs in VK2/E6E7 cells. Cells were incubated with PBS, non-silencing siRNA PLGA-PEG NP (1.334 mg/mL), non-silencing siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) or PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL) and autophagy inhibitor (bafilomycin A, 50 mM), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) and autophagy inducer (rapamycin, 100 nM) for 48 hr and then infected with C. trachomatis. Images were taken 24 hr post C. trachomatis infection. ( A ) Intracellular C. trachomatis RB foci (green fluorescence) were visualized using fluorescence microscopy. Experiments were conducted n = 3 and a group of representative images were shown. ( B ) Semi-quantitative measurements of intracellular C. trachomatis RB foci were accomplished using Image J software. Values represent the mean ± SD, n = 3. *p

    Article Snippet: Cell culture and C. trachomatis propagation Vaginal epithelial cells (VK2/E6E7), C. trachomatis strain K, and McCoy cells were purchased from ATCC (Virginia, USA).

    Techniques: Incubation, Infection, Fluorescence, Microscopy, Software

    In vitro quantitation of C. trachomatis genomes in the supernatant of VK2/E6E7 cell culture. Cells were incubated with PBS, non-silencing siRNA PLGA-PEG NP (1.334 mg/mL), non-silencing siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) or PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL) and autophagy inhibitor (bafilomycin A, 50 mM), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) and autophagy inducer (rapamycin, 100 nM) for 48 hr and then infected with C. trachomatis. Supernatant containing newly produced C. trachomatis genomes was collected 24 hr post C. trachomatis infection and quantified by qRT-PCR. Values represent the mean ± SD, n = 3. *p

    Journal: Scientific Reports

    Article Title: Autophagy induction and PDGFR-β knockdown by siRNA-encapsulated nanoparticles reduce chlamydia trachomatis infection

    doi: 10.1038/s41598-018-36601-y

    Figure Lengend Snippet: In vitro quantitation of C. trachomatis genomes in the supernatant of VK2/E6E7 cell culture. Cells were incubated with PBS, non-silencing siRNA PLGA-PEG NP (1.334 mg/mL), non-silencing siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) or PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL), PDGFR-β siRNA-PEI-PLGA-PEG NP (1.334 mg/mL) and autophagy inhibitor (bafilomycin A, 50 mM), PDGFR-β siRNA PLGA-PEG NP (1.334 mg/mL) and autophagy inducer (rapamycin, 100 nM) for 48 hr and then infected with C. trachomatis. Supernatant containing newly produced C. trachomatis genomes was collected 24 hr post C. trachomatis infection and quantified by qRT-PCR. Values represent the mean ± SD, n = 3. *p

    Article Snippet: Cell culture and C. trachomatis propagation Vaginal epithelial cells (VK2/E6E7), C. trachomatis strain K, and McCoy cells were purchased from ATCC (Virginia, USA).

    Techniques: In Vitro, Quantitation Assay, Cell Culture, Incubation, Infection, Produced, Quantitative RT-PCR

    Cytokine production by VK2 cells in response to Gardnerella vaginalis in the presence or absence of clinical Lactobacillus isolates (n = 16). Immortalized VK2 cells were cultured to confluence and then treated with Lactobacillus isolates adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media before being incubated for 5 hours at 37 °C with 5% CO 2 . G. vaginalis cultures at a concentration of 1 × 10 7 CFU/ml were then added and incubated for a further 20 hours. Cytokine concentrations were measured in the culture supernatants using Luminex. Mann Whitney U tests were used to compare cytokine responses and p-values were adjusted for multiple comparisons using a false discovery rate step down procedure. ( A ) Data are presented as Tukey box plots. Boxes represent the interquartile ranges, lines within boxes represent medians and whiskers represent minimum and maximum values. *Adjusted p-values

    Journal: Scientific Reports

    Article Title: Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota

    doi: 10.1038/s41598-020-62184-8

    Figure Lengend Snippet: Cytokine production by VK2 cells in response to Gardnerella vaginalis in the presence or absence of clinical Lactobacillus isolates (n = 16). Immortalized VK2 cells were cultured to confluence and then treated with Lactobacillus isolates adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media before being incubated for 5 hours at 37 °C with 5% CO 2 . G. vaginalis cultures at a concentration of 1 × 10 7 CFU/ml were then added and incubated for a further 20 hours. Cytokine concentrations were measured in the culture supernatants using Luminex. Mann Whitney U tests were used to compare cytokine responses and p-values were adjusted for multiple comparisons using a false discovery rate step down procedure. ( A ) Data are presented as Tukey box plots. Boxes represent the interquartile ranges, lines within boxes represent medians and whiskers represent minimum and maximum values. *Adjusted p-values

    Article Snippet: Vaginal epithelial cell stimulation and measurement of cytokine concentrations Vaginal epithelial cells (VK2/E6E7 ATCC CRL-2616), that closely resemble the tissue of origin, were maintained in complete keratinocyte serum free media (KSFM) supplemented with 0.4 mM calcium chloride, 0.05 mg/ml of bovine pituitary extract, 0.1 ng/ml human recombinant epithelial growth factor and 50 U/ml penicillin and 50 U/ml streptomycin (Sigma-Aldrich, St. Louis, Missouri) as described previously.

    Techniques: Cell Culture, Incubation, Concentration Assay, Luminex, MANN-WHITNEY

    ( A ) Gram stained images of Lactobacillus adhesion to VK2 cells. Lactobacillus isolates (n = 64) were cultured and adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media before being added to VK2 cell monolayers in chamber slides and incubated for 2 hours at 37 °C with 5% CO 2 . Non-adherent bacteria were removed with sterile phosphate buffered saline (PBS) before the slides were Gram stained. Representative images of the Gram stained slides were collected and Lactobacillus isolates were ranked according to level of adhesion in ascending order from least adherent ( 1 ) to the most adherent ( 6 ). Level of adhesion to VK2 cells, growth rates and lengths of Lactobacillus isolates obtained from women with optimal [Nugent score: 0–3 (n = 36)] and non-optimal microbiota [Nugent score: 4–10 (n = 28)]. ( B ) Adhesion was determined by adding Lactobacillus cultures adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media to VK2 cell monolayers and incubating for 2 hours at 37 °C with 5% CO 2 . Non-adherent bacteria were removed with sterile phosphate buffered saline (PBS) before the slides were Gram stained. Each isolate was then scored according to level of adhesion (1–6) by two individuals blinded to the cytokine profiles of the isolates. ( C ) Growth rates were evaluated by measuring the optical densities at a wavelength of 600 nm, of Lactobacillus cultures initially adjusted to 4.18 × 10 6 CFU/ml and incubated in de Man Rogosa and Sharpe (MRS) broth anaerobically for 24 hours. The areas under the curve were determined during the active phase of growth. ( D ) Relative bacterial size. Single colonies were picked from Lactobacillus cultures (n = 64) and smears were prepared on microscope slides and Gram-stained before taking images at 1,000x magnification. Bacterial length was determined from the images using Image J software. The mean of five measurements for each isolate was used for analysis. Boxes represent the interquartile ranges, lines within boxes represent medians and whiskers represent minimum and maximum values. P-values

    Journal: Scientific Reports

    Article Title: Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota

    doi: 10.1038/s41598-020-62184-8

    Figure Lengend Snippet: ( A ) Gram stained images of Lactobacillus adhesion to VK2 cells. Lactobacillus isolates (n = 64) were cultured and adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media before being added to VK2 cell monolayers in chamber slides and incubated for 2 hours at 37 °C with 5% CO 2 . Non-adherent bacteria were removed with sterile phosphate buffered saline (PBS) before the slides were Gram stained. Representative images of the Gram stained slides were collected and Lactobacillus isolates were ranked according to level of adhesion in ascending order from least adherent ( 1 ) to the most adherent ( 6 ). Level of adhesion to VK2 cells, growth rates and lengths of Lactobacillus isolates obtained from women with optimal [Nugent score: 0–3 (n = 36)] and non-optimal microbiota [Nugent score: 4–10 (n = 28)]. ( B ) Adhesion was determined by adding Lactobacillus cultures adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media to VK2 cell monolayers and incubating for 2 hours at 37 °C with 5% CO 2 . Non-adherent bacteria were removed with sterile phosphate buffered saline (PBS) before the slides were Gram stained. Each isolate was then scored according to level of adhesion (1–6) by two individuals blinded to the cytokine profiles of the isolates. ( C ) Growth rates were evaluated by measuring the optical densities at a wavelength of 600 nm, of Lactobacillus cultures initially adjusted to 4.18 × 10 6 CFU/ml and incubated in de Man Rogosa and Sharpe (MRS) broth anaerobically for 24 hours. The areas under the curve were determined during the active phase of growth. ( D ) Relative bacterial size. Single colonies were picked from Lactobacillus cultures (n = 64) and smears were prepared on microscope slides and Gram-stained before taking images at 1,000x magnification. Bacterial length was determined from the images using Image J software. The mean of five measurements for each isolate was used for analysis. Boxes represent the interquartile ranges, lines within boxes represent medians and whiskers represent minimum and maximum values. P-values

    Article Snippet: Vaginal epithelial cell stimulation and measurement of cytokine concentrations Vaginal epithelial cells (VK2/E6E7 ATCC CRL-2616), that closely resemble the tissue of origin, were maintained in complete keratinocyte serum free media (KSFM) supplemented with 0.4 mM calcium chloride, 0.05 mg/ml of bovine pituitary extract, 0.1 ng/ml human recombinant epithelial growth factor and 50 U/ml penicillin and 50 U/ml streptomycin (Sigma-Aldrich, St. Louis, Missouri) as described previously.

    Techniques: Staining, Cell Culture, Incubation, Microscopy, Software

    Cytokine production by vaginal epithelial (VK2) cells in response to vaginal Lactobacillus isolates. ( A ) Heatmap of log 10 -transformed concentrations of cytokines produced by VK2 cells stimulated with Lactobacillus isolates (n = 64) obtained from women with optimal (n = 36), intermediate (n = 8) and non-optimal microbiota (n = 20). Lactobacillus cultures were adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media then added to VK2 cell monolayers before being incubated for 24 hours at 37 °C with 5% CO 2 . Cytokine concentrations in the cell culture supernatants were measured using Luminex. Level of adhesion to VK2 cells, bacterial vaginosis (BV) status, and Lactobacillus species are also shown on the left side of the heatmap. ( B ) Inflammatory cytokine production in response to Lactobacillus isolates from women with optimal microbiota (Nugent 0–3; n = 36), including L. crispatus (n = 6); L. jensenii (n = 12), L. johnsonii (n = 5), L. mucosae (n = 4), L. plantarum (n = 1), L. vaginalis (n = 8), compared to women with non-optimal microbiota (Nugent 4–10; n = 28), including L. crispatus (n = 5), L. jensenii (n = 2), L. mucosae (n = 11), L. plantarum (n = 1), L. ruminis (n = 5), L. salivarius (n = 2) and L. vaginalis (n = 2). Data are shown as Tukey box plots. Boxes represent the interquartile ranges, lines within boxes represent medians and whiskers represent minimum and maximum values. P-values were adjusted for multiple comparisons using a false discovery rate step down procedure. *Adjusted p-values

    Journal: Scientific Reports

    Article Title: Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota

    doi: 10.1038/s41598-020-62184-8

    Figure Lengend Snippet: Cytokine production by vaginal epithelial (VK2) cells in response to vaginal Lactobacillus isolates. ( A ) Heatmap of log 10 -transformed concentrations of cytokines produced by VK2 cells stimulated with Lactobacillus isolates (n = 64) obtained from women with optimal (n = 36), intermediate (n = 8) and non-optimal microbiota (n = 20). Lactobacillus cultures were adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media then added to VK2 cell monolayers before being incubated for 24 hours at 37 °C with 5% CO 2 . Cytokine concentrations in the cell culture supernatants were measured using Luminex. Level of adhesion to VK2 cells, bacterial vaginosis (BV) status, and Lactobacillus species are also shown on the left side of the heatmap. ( B ) Inflammatory cytokine production in response to Lactobacillus isolates from women with optimal microbiota (Nugent 0–3; n = 36), including L. crispatus (n = 6); L. jensenii (n = 12), L. johnsonii (n = 5), L. mucosae (n = 4), L. plantarum (n = 1), L. vaginalis (n = 8), compared to women with non-optimal microbiota (Nugent 4–10; n = 28), including L. crispatus (n = 5), L. jensenii (n = 2), L. mucosae (n = 11), L. plantarum (n = 1), L. ruminis (n = 5), L. salivarius (n = 2) and L. vaginalis (n = 2). Data are shown as Tukey box plots. Boxes represent the interquartile ranges, lines within boxes represent medians and whiskers represent minimum and maximum values. P-values were adjusted for multiple comparisons using a false discovery rate step down procedure. *Adjusted p-values

    Article Snippet: Vaginal epithelial cell stimulation and measurement of cytokine concentrations Vaginal epithelial cells (VK2/E6E7 ATCC CRL-2616), that closely resemble the tissue of origin, were maintained in complete keratinocyte serum free media (KSFM) supplemented with 0.4 mM calcium chloride, 0.05 mg/ml of bovine pituitary extract, 0.1 ng/ml human recombinant epithelial growth factor and 50 U/ml penicillin and 50 U/ml streptomycin (Sigma-Aldrich, St. Louis, Missouri) as described previously.

    Techniques: Transformation Assay, Produced, Incubation, Cell Culture, Luminex

    Inflammatory cytokine production by VK2 cells in response to different vaginal Lactobacillus species. Lactobacillus isolates, including L. crispatus (n = 11), L. jensenii (n = 14), L. johnsonii (n = 5), L. mucosae (n = 15), L. plantarum (n = 2), L. ruminis (n = 5), L. salivarius (n = 2) and L. vaginalis (n = 10), were adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media before being incubated with VK2 cells for 24 hours at 37 °C with 5% CO 2 . Cytokine concentrations were measured in the culture supernatants using Luminex. ( A ) Stacked bars showing median concentrations of each cytokine interleukin (IL)-1α, IL-1β, IL-6, IL-8, IFN-γ-inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-3α and regulatory IL-1 receptor antagonist (RA). ( B ) Stacked bars showing median concentrations of each cytokine excluding IL-1RA (IL-1α, IL-1β, IL-6, IL-8, IP-10, MIP-1α, MIP-1β and MIP-3α).

    Journal: Scientific Reports

    Article Title: Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota

    doi: 10.1038/s41598-020-62184-8

    Figure Lengend Snippet: Inflammatory cytokine production by VK2 cells in response to different vaginal Lactobacillus species. Lactobacillus isolates, including L. crispatus (n = 11), L. jensenii (n = 14), L. johnsonii (n = 5), L. mucosae (n = 15), L. plantarum (n = 2), L. ruminis (n = 5), L. salivarius (n = 2) and L. vaginalis (n = 10), were adjusted to 4.18 × 10 6 colony forming units (CFU)/ml in antibiotic free keratinocyte serum free media before being incubated with VK2 cells for 24 hours at 37 °C with 5% CO 2 . Cytokine concentrations were measured in the culture supernatants using Luminex. ( A ) Stacked bars showing median concentrations of each cytokine interleukin (IL)-1α, IL-1β, IL-6, IL-8, IFN-γ-inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-3α and regulatory IL-1 receptor antagonist (RA). ( B ) Stacked bars showing median concentrations of each cytokine excluding IL-1RA (IL-1α, IL-1β, IL-6, IL-8, IP-10, MIP-1α, MIP-1β and MIP-3α).

    Article Snippet: Vaginal epithelial cell stimulation and measurement of cytokine concentrations Vaginal epithelial cells (VK2/E6E7 ATCC CRL-2616), that closely resemble the tissue of origin, were maintained in complete keratinocyte serum free media (KSFM) supplemented with 0.4 mM calcium chloride, 0.05 mg/ml of bovine pituitary extract, 0.1 ng/ml human recombinant epithelial growth factor and 50 U/ml penicillin and 50 U/ml streptomycin (Sigma-Aldrich, St. Louis, Missouri) as described previously.

    Techniques: Incubation, Luminex

    Internalization of VEC cells by G. vaginalis . VK2 vaginal epithelial cells were exposed to G. vaginalis for 24 h. Cells were then fixed and dual stained for estrogen receptor beta (rhodamine) and G. vaginalis (FITC). DAPI was used to stain nuclei blue.

    Journal: Microbes and infection / Institut Pasteur

    Article Title: Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

    doi: 10.1016/j.micinf.2011.12.009

    Figure Lengend Snippet: Internalization of VEC cells by G. vaginalis . VK2 vaginal epithelial cells were exposed to G. vaginalis for 24 h. Cells were then fixed and dual stained for estrogen receptor beta (rhodamine) and G. vaginalis (FITC). DAPI was used to stain nuclei blue.

    Article Snippet: Human immortalized VK2 cells (ATCC 2616), obtained from the American Type Culture Collection, were selected for use because these cells represent squamous vaginal epithelial cells that are sloughed off and contained in vaginal discharges used to clinically identify BV [ ].

    Techniques: Staining

    Transmission electron microscopy (TEM) of VK2 cells exposed to G. vaginalis for 1 h. Bacterial cell dimensions are given in nanometers (arrows and white text). The bars at the bottom of B–D represent length in nanometers.(A). G. vaginalis bacteria

    Journal: Microbes and infection / Institut Pasteur

    Article Title: Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

    doi: 10.1016/j.micinf.2011.12.009

    Figure Lengend Snippet: Transmission electron microscopy (TEM) of VK2 cells exposed to G. vaginalis for 1 h. Bacterial cell dimensions are given in nanometers (arrows and white text). The bars at the bottom of B–D represent length in nanometers.(A). G. vaginalis bacteria

    Article Snippet: Human immortalized VK2 cells (ATCC 2616), obtained from the American Type Culture Collection, were selected for use because these cells represent squamous vaginal epithelial cells that are sloughed off and contained in vaginal discharges used to clinically identify BV [ ].

    Techniques: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    3.1. Phenotypic characterization of VK2 cells

    Journal: Microbes and infection / Institut Pasteur

    Article Title: Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

    doi: 10.1016/j.micinf.2011.12.009

    Figure Lengend Snippet: 3.1. Phenotypic characterization of VK2 cells

    Article Snippet: Human immortalized VK2 cells (ATCC 2616), obtained from the American Type Culture Collection, were selected for use because these cells represent squamous vaginal epithelial cells that are sloughed off and contained in vaginal discharges used to clinically identify BV [ ].

    Techniques:

    Invasion assays using 35 S labeled bacteria and VK2 cells ± cytochalasin-D. (A). VK2 cells exposed separately to 35 S labeled G. vaginalis , heat-killed G. vaginalis , L. acidophilus and L. crispatus for 1 h, using either KSFM or calcium-free KSFM

    Journal: Microbes and infection / Institut Pasteur

    Article Title: Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

    doi: 10.1016/j.micinf.2011.12.009

    Figure Lengend Snippet: Invasion assays using 35 S labeled bacteria and VK2 cells ± cytochalasin-D. (A). VK2 cells exposed separately to 35 S labeled G. vaginalis , heat-killed G. vaginalis , L. acidophilus and L. crispatus for 1 h, using either KSFM or calcium-free KSFM

    Article Snippet: Human immortalized VK2 cells (ATCC 2616), obtained from the American Type Culture Collection, were selected for use because these cells represent squamous vaginal epithelial cells that are sloughed off and contained in vaginal discharges used to clinically identify BV [ ].

    Techniques: Labeling

    Cytotoxicity of GML and DDG to Human Vaginal Epithelial Cells (HVECs). HVECs were exposed to GML (▪) and DDG (▴) for 6 h. Cytotoxicity was accessed by measuring the release of LDH. Error bars are SEM. The dashed line indicates median cell survival (LD 50 ). Symbols:▪, GML; ▴, DDG.

    Journal: PLoS ONE

    Article Title: Glycerol Monolaurate and Dodecylglycerol Effects on Staphylococcus aureus and Toxic Shock Syndrome Toxin-1 In Vitro and In Vivo

    doi: 10.1371/journal.pone.0007499

    Figure Lengend Snippet: Cytotoxicity of GML and DDG to Human Vaginal Epithelial Cells (HVECs). HVECs were exposed to GML (▪) and DDG (▴) for 6 h. Cytotoxicity was accessed by measuring the release of LDH. Error bars are SEM. The dashed line indicates median cell survival (LD 50 ). Symbols:▪, GML; ▴, DDG.

    Article Snippet: Cytotoxicity of the compounds to human vaginal cells Immortalized human vaginal epithelial cells (ATCC CRL-2616) were used to determine mammalian cell cytotoxicity of GML and DDG.

    Techniques:

    Bisulfite sequencing analysis of the DEFB1 promoter region in VK2/E6E7 cells. a The schematic representation of the 5′ end of DEFB1 is marked by the typical non-CpG island promoter. The arrow indicates the transcription start site (TSS), and the gray box represents the first exon. CpG loci and the relative positions of primers for bisulfite sequencing are indicated under the CpG island plot. b Bisulfite sequencing of the DEFB1 promoter, which is divided into “proximal” and “distal” regions, was performed on VK2/E6E7 cells treated with DAC or L. gasseri . A single nucleotide polymorphism present in the CpG 8 locus within the DEFB1 promoter region is denoted by the symbol x . Circles represent single CpG sites, and each row of circles represents the DNA sequence of an individual clone. Unmethylated and methylated CpG sites are depicted as white and black circles , respectively. The red boxes highlight the promoter regions spanning the key CpG loci that are important for the difference in the DEFB1 expression in DAC-treated VK2/E6E7 cells compared to untreated control cells

    Journal: Probiotics and Antimicrobial Proteins

    Article Title: Distinct Histone Modifications Modulate DEFB1 Expression in Human Vaginal Keratinocytes in Response to Lactobacillus spp.

    doi: 10.1007/s12602-017-9286-6

    Figure Lengend Snippet: Bisulfite sequencing analysis of the DEFB1 promoter region in VK2/E6E7 cells. a The schematic representation of the 5′ end of DEFB1 is marked by the typical non-CpG island promoter. The arrow indicates the transcription start site (TSS), and the gray box represents the first exon. CpG loci and the relative positions of primers for bisulfite sequencing are indicated under the CpG island plot. b Bisulfite sequencing of the DEFB1 promoter, which is divided into “proximal” and “distal” regions, was performed on VK2/E6E7 cells treated with DAC or L. gasseri . A single nucleotide polymorphism present in the CpG 8 locus within the DEFB1 promoter region is denoted by the symbol x . Circles represent single CpG sites, and each row of circles represents the DNA sequence of an individual clone. Unmethylated and methylated CpG sites are depicted as white and black circles , respectively. The red boxes highlight the promoter regions spanning the key CpG loci that are important for the difference in the DEFB1 expression in DAC-treated VK2/E6E7 cells compared to untreated control cells

    Article Snippet: Human Cell and Bacterial Cultures The human vaginal keratinocyte VK2/E6E7 cell line (ATCC CRL-261) was obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Methylation Sequencing, Sequencing, Methylation, Expressing

    Significant changes in DEFB1 expression in vaginal keratinocytes VK2/E6E7 after treatment with either epigenetic inhibitors or Lactobacillus spp. a Quantitative RT-PCR analysis showed the induction of DEFB1 expression upon treatment with the DNA-demethylating agent 2′-deoxy-5-azacytidine (DAC) or the HDAC inhibitor trichostatin A (TSA). b Up- or down-regulation of the DEFB1 mRNA level in response to exposure to either L. gasseri or L. reuteri . After normalization of DEFB1 expression by use of the HPRT1 mRNA level, the relative expression level of DEFB1 in lactobacilli-treated cells relative to that in untreated control cells was calculated. * P

    Journal: Probiotics and Antimicrobial Proteins

    Article Title: Distinct Histone Modifications Modulate DEFB1 Expression in Human Vaginal Keratinocytes in Response to Lactobacillus spp.

    doi: 10.1007/s12602-017-9286-6

    Figure Lengend Snippet: Significant changes in DEFB1 expression in vaginal keratinocytes VK2/E6E7 after treatment with either epigenetic inhibitors or Lactobacillus spp. a Quantitative RT-PCR analysis showed the induction of DEFB1 expression upon treatment with the DNA-demethylating agent 2′-deoxy-5-azacytidine (DAC) or the HDAC inhibitor trichostatin A (TSA). b Up- or down-regulation of the DEFB1 mRNA level in response to exposure to either L. gasseri or L. reuteri . After normalization of DEFB1 expression by use of the HPRT1 mRNA level, the relative expression level of DEFB1 in lactobacilli-treated cells relative to that in untreated control cells was calculated. * P

    Article Snippet: Human Cell and Bacterial Cultures The human vaginal keratinocyte VK2/E6E7 cell line (ATCC CRL-261) was obtained from the American Type Culture Collection (Manassas, VA, USA).

    Techniques: Expressing, Quantitative RT-PCR