human vec line vk2 e6 e7  (ATCC)

Journal: Nature Communications

Article Title: Invasion of vaginal epithelial cells by uropathogenic Escherichia coli

doi: 10.1038/s41467-020-16627-5

Figure Lengend Snippet: VK2 E6/E7 cells were infected with the indicated strain of UPEC. a and b Gentamicin-based adherence and invasion assay of VECs infected by: a UTI89 at the indicated MOIs for 30 min. b UTI89 at a MOI of 5:1 for 30, 120, and 240 min. Planktonic bacteria are washed away by PBS, which leaves behind the adherent bacteria. Gentamicin kills extracellular bacteria; whereas, bacteria that invade cells are safe from the membrane impermeable antibiotic. UTI89 and clinical isolates adhered to and invaded VECs to different extents. Adherent (medium gray bars) and intracellular (light gray bars) bacterial populations are relative to the average of the total population of bacteria (dark gray bars) within the well. a , b Data for initially setting-up the model are presented as the mean of nine independent experiments with standard deviation to demonstrate the level of consistency. c Confocal laser scanning microscopy images of mock infected (PBS) and UTI89/pCom-GFP for 30, 120, and 240 min that were stained with ToPro-3 for DNA and r-WGA to outline VEC membranes. d UTI89 at a MOI of 50:1 for 2 h imaged by TEM. TEM images are representative of two independent experiments. e Microscopy image of VK2 E6/E7 cells infected with UTI89/pCom-GFP for 120 min were stained with r-WGA; additionally, to further differentiate E. coli localization permeabilization was not performed and extracellular E. coli cells were stained with α- E. coli (blue). White arrows point toward intracellular bacteria. Confocal laser scanning microscopy images are representative of three independent experiments. f Low-passage clinical UPEC isolates were utilized in gentamicin-based adherence (medium gray bars) and invasion (light gray bars) assay with VECs for 2 h. f Data are the mean of nine independent experiments with error bars representing s.e.m. For statistical analysis two-way ANOVA with Tukey post-hoc test (* P < 0.05, ** P < 0.01).

Article Snippet: The human VEC line VK2 E6/E7 (ATCC CRL-2616) were cultured in keratinocyte-serum-free media (KSFM) (Life Technologies Co., Grand Island, NY) supplemented with 0.1 ng/mL human recombinant epidermal growth factor, 0.05 mg/mL bovine pituitary extract, and 0.4 mM calcium chloride .

Techniques: Infection, Invasion Assay, Bacteria, Membrane, Standard Deviation, Confocal Laser Scanning Microscopy, Staining, Microscopy

Journal: Nature Communications

Article Title: Invasion of vaginal epithelial cells by uropathogenic Escherichia coli

doi: 10.1038/s41467-020-16627-5

Figure Lengend Snippet: a – h Adherence and invasion assays were performed at a MOI of 5:1 for 2 h. Adherent (medium gray bars) and intracellular (light gray bars) bacterial populations are relative to the average of the total population of bacteria (dark gray bars) within the well. a Adherence and invasion assay comparison of UTI89 and Δ fimA-H strains on VK2 E6/E7 cells and b comparison of UTI89 on 5637 bladder and VK2 E2/E7 vaginal cell lines. Data are the mean of nine independent experiments with error bars representing s.e.m. For statistical analysis two-way ANOVA with Sidak’s multiple comparisons test (* P = 0.0402). c Adherent and invasive UTI89/pCom-GFP associated with VK2 E6/E7 with stained by phalloidin conjugated with tetramethylrhodamine, for F-actin and ToPro-3. Red bundles specifically around bacteria indicated actin polymerization occurs prior to invasion. Experimental controls include PFA-killed UTI89/pCom-GFP and latex beads. d – i Invasion as determined by gentamicin-based invasion assay with UTI89 with the indicated drug. d F-actin inhibitor, cytochalasin D, reversibly inhibited UTI89 invasion into VECs. e Genistein, a specific inhibitor of protein tyrosine kinases inhibited UTI89 VEC invasion. f Wortmannin, a potent inhibitor of PI3K, minorly reduced UTI89 VEC invasion. g PP1, an inhibitor of Src-family kinases, inhibited VEC invasion in a dose-dependent manner. h Three different inhibitors of microtubules: nocodazole, taxol, and vinblastine diminished UTI89 VEC invasion. i Three histone deacetylase inhibitors trichostatin A, nicotinamide, and butyrate do not prevent UTI89 from invading VECs. Percent invasion is relative to the control group (DMSO). Data are representative of the mean of nine independent experiments with error bars representing s.e.m. d – h For statistical analysis non-parametric Kruskal–Wallis with two-sided Dunn’s post-hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). j SEM images of UTI89 and Δ fimA-H , type 1 pili mutant strain, interacting with the surface of VK2 E6/E7 cells. Microfolds and microvilli are typical of VECs. SEM images of the subtle envelopment of UTI89 by VEC membranes is consistent with a zipper-like model of invasion. Images are representative of three independent experiments.

Article Snippet: The human VEC line VK2 E6/E7 (ATCC CRL-2616) were cultured in keratinocyte-serum-free media (KSFM) (Life Technologies Co., Grand Island, NY) supplemented with 0.1 ng/mL human recombinant epidermal growth factor, 0.05 mg/mL bovine pituitary extract, and 0.4 mM calcium chloride .

Techniques: Bacteria, Invasion Assay, Comparison, Staining, Histone Deacetylase Assay, Control, Mutagenesis

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on VEC viability . VK2/E6E7 cells were treated with rhIFNα-2b for 24 h. The mean values for the remaining four values and standard deviations (error bars) are shown. ∗∗∗ , significant difference compared to 0 mg/mL of rhIFNα-2b control group ( P < 0.0001).

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Control

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on the production of IL-2 (A) , IL-4 (B) , IL-6 (C) , IL-8 (D) , and IL-17 (E) (expressed as pg/mL) by the VEC line, VK2/E6E7 cells cultivated alone, grown with 1.25 mg/mL rhIFNα-2b, and infected with C. albicans (1 × 10 5 /mL). The supernatants were collected and the cytokine levels were assessed by performing an ELISA 12 h post-infection and a subsequent 24 h of co-incubation with 1.25 mg/mL of rhIFNα-2b. V represents the VECs cultivated alone; V+I represents VECs co-incubated with 1.25 mg/mL of rhIFNα-2b for 24 h; V+C represents VECs infected with Candida albicans for 12 h; V+C+I represents the VECs infected with C. albicans for 12 h, then treated with 1.25 mg/mL of rhIFNα-2b for another 24 h. ∗∗ , significant difference compared to the V group ( P < 0.001); ∗∗∗ , significant difference compared to the V group ( P < 0.0001).

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on the production of vaginal epithelial-derived IgG (expressed in μg/mL) by the VEC line, VK2/E6E7 cells . Supernatants were collected, and the IgG levels were assessed by performing an ELISA after 12 h of infection and a subsequent 24 h of co-incubation with rhIFNα-2b. V represents the VECs cultivated alone; V+I represents the VECs co-incubated with 1.25 mg/mL of rhIFNα-2b for 24 h; V+C represents the VECs infected with C. albicans for 12 h; V+C+I represents the VECs infected with C. albicans for 12 h, then treated with 1.25 mg/mL of rhIFNα-2b for another 24 h. ∗∗∗ , significant difference compared to the V group ( P < 0.0001); ∗∗ , significant difference compared to the V+C group ( P = 0.004). Each sample was repeated three times. The error bars indicate the standard deviation.

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on VEC-mediated anti-Candida activity . SEM of the control cells (A) , C. albicans infected cells at 12 h (B) , and rhIFNα-2b treated cells (C,D) . (C,D) represent the VECs infected with C. albicans for 12 h, then treated with 1.25 mg/mL rhIFNα-2b for another 24 h. Microvilli are indicated by small red arrows, filopodia are indicated with a small yellow arrow, pseudohyphae are indicated with a small blue arrow, and living VK2 cells indicated with white arrows.

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Activity Assay, Control, Infection