Journal: PLOS One

Article Title: A novel peptide mimetic, brilacidin, for combating multidrug-resistant Neisseria gonorrhoeae

doi: 10.1371/journal.pone.0325722

Figure Lengend Snippet: A) Propidium iodide fluorescence after treating N. gonorrhoeae with either azithromycin or brilacidin to predict the permeabilization of the cytoplasmic membrane. B) Percentage of ATP leakage from N. gonorrhoeae treated with brilacidin or azithromycin relative to Triton X-100 (positive control with complete ATP leakage). Asterisks denote statistically significant differences between test agents and DMSO (untreated), * (P < 0.05), ** (P < 0.01), and **** (P < 0.0001) as determined by one-way ANOVA.

Article Snippet: Media and reagents including McCoy’s 5A medium and hematin (Sigma Aldrich, St. Louis, MO, USA), triton X-100 (Acros Organics, Fair Lawn, NJ, USA), BacTiter-Glo reagent (Promega Corporation, Madison, WI, USA), Propidium iodide (PI) and nicotinamide adenine dinucleotide (NAD) (Chem-Impex International, Wood Dale, IL, USA), MTS (3-(4,5-dimethylthia- zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Abcam, Waltham, MA, USA), and brucella broth, chocolate II agar plates, IsoVitaleX and bovine hemoglobin (Becton, Dickinson and Company, Cockeysville, MD, USA), were obtained from chemical vendors.

Techniques: Fluorescence, Membrane, Positive Control

Journal: Cancers

Article Title: NGFI-A Binding Protein 2 Promotes EGF-Dependent HNSCC Cell Invasion

doi: 10.3390/cancers11030315

Figure Lengend Snippet: Effect of NAB2 on EGF-dependent head and neck squamous cell carcinoma (HNSCC) cell invasion. ( A ) NAB2 mRNA and protein expression following EGF treatment (50 ng/mL) for 24 h was analyzed in FaDU cells by qPCR and Western blot analysis. ( B ) FaDU cells were transfected with an siNAB2 mixture (40 nm/mL) for 48 h, after which the mRNA and protein expression of NAB2 were analyzed. ( C ) Two-dimensional Transwell Matrigel invasion assays were performed to analyze the effect of NAB2 knockdown, followed by EGF treatment, on FaDU cell invasion. Control siRNA- or siNAB2-transfected FaDU cells were added to each Matrigel-coated Transwell. After culturing for 48 h, the cells were washed twice with phosphate-bufferd saline and stained with 0.2% crystal violet in 10% ethanol (5× magnification). The cell invasion index was calculated as the difference between the number of invading cells in the siNAB2-transfected group compared to that in the control siRNA-transfected group (* p < 0.01). ( D ) Matrigel invasion assays using YD-10B cells were performed with the same conditions (5× magnification) (** p < 0.005). The results shown are from two or three independent experiments, with each bar representing the standard deviation.

Article Snippet: Cells were suspended in serum-free medium, which was followed by transfection with control siRNA (sc-37007) or an siNAB2 mixture (sc-36014) consisting of three specific targeting oligonucleotides (Santa Cruz Biotechnology) using an electroporation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Techniques: Expressing, Western Blot, Transfection, Knockdown, Control, Saline, Staining, Standard Deviation

Journal: Cancers

Article Title: NGFI-A Binding Protein 2 Promotes EGF-Dependent HNSCC Cell Invasion

doi: 10.3390/cancers11030315

Figure Lengend Snippet: Effect of NAB2 on EGF-dependent FaDU spheroid invasion. ( A ) FaDU cells were seeded in a 96-well ultralow attachment U-bottom plate (6000 cells per well) and cultured for 4 days to form one spheroid per well (>700 μm in diameter). Control siRNA or siNAB2 was transfected into FaDU spheroids using Lipofectamine 2000 for 10 days and NAB2 mRNA expression was evaluated by qPCR. ( B ) The Matrigel invasion of FaDU spheroids transfected with siNAB2 was investigated after EGF treatment (50 ng/mL). FaDU spheroids cultured for 5 days were transfected with control siRNA or siNAB2 using Lipofectamine 2000. After 8 h, the spheroids were centrifuged and 50 μL of Matrigel matrix was added directly to each well (100 μL of medium) to provide a semi-solid matrix into which tumor cells could migrate from the spheroid body. Matrigel invasion was monitored over a period of 10 days by phase-contrast microscopy (5× magnification). ( C ) Matrigel invasion was quantified based on the average length of tube-like structures protruding from the surface of each spheroid (* p < 0.005). The results shown are from two or three independent experiments, with each bar representing the standard deviation.

Article Snippet: Cells were suspended in serum-free medium, which was followed by transfection with control siRNA (sc-37007) or an siNAB2 mixture (sc-36014) consisting of three specific targeting oligonucleotides (Santa Cruz Biotechnology) using an electroporation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Techniques: Cell Culture, Control, Transfection, Expressing, Microscopy, Standard Deviation

Journal: Cancers

Article Title: NGFI-A Binding Protein 2 Promotes EGF-Dependent HNSCC Cell Invasion

doi: 10.3390/cancers11030315

Figure Lengend Snippet: Effect of knockdown or overexpression of EGR1, NAB2, and Sp1 on MMP expression. ( A ) FaDU cells were transfected with control siRNA or siNAB2 for 24 h, and then subjected to EGF treatment (50 ng/mL) for another 24 h. Transfected cells were analyzed for mRNA and protein expression of MMP2 and MMP9. Protein expression was quantified and plotted in three independent experiments. ( B ) A schematic representation of the promoter regions containing the consensus EGR1/Sp1-binding sites (black arrowheads). FaDU cells were transfected with control siRNA or siNAB2 and chromatin was immunoprecipitated with ( C ) anti-Sp1 antibody or ( D ) anti-EGR1 antibody. The resulting immunoprecipitates were analyzed by qPCR to detect the consensus promoter sequences. ( E ) EGR1 / NAB2 genes were overexpressed in FaDU cells, after which chromatin was immunoprecipitated with an anti-Sp1 antibody. The resulting immunoprecipitates were analyzed by qPCR to detect the consensus promoter sequences. ( F ) For luciferase reporter assays using MMP2-1 and MMP9-1, promoter fragments were synthesized and cloned into the pGL4.70 vector. After 24 h of vector transfection, dual luciferase assays were performed to determine the effect of different combinations of EGR1/NBA2 overexpression (over) and/or Sp1 knockdown. The results shown are based on two or three independent experiments, with each bar representing the standard deviation.

Article Snippet: Cells were suspended in serum-free medium, which was followed by transfection with control siRNA (sc-37007) or an siNAB2 mixture (sc-36014) consisting of three specific targeting oligonucleotides (Santa Cruz Biotechnology) using an electroporation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Techniques: Knockdown, Over Expression, Expressing, Transfection, Control, Binding Assay, Immunoprecipitation, Luciferase, Synthesized, Clone Assay, Plasmid Preparation, Standard Deviation