Journal: Journal of Virology

Article Title: CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling

doi: 10.1128/JVI.02251-16

Figure Lengend Snippet: LMP1 trafficking to lipid rafts and downstream signaling do not require CD63. (A) HEK293 control and CD63 knockout cells were transfected with GFP-LMP1. Lipid rafts were biochemically isolated for immunoblot analysis of whole-cell (input) and lipid raft-associated proteins, equal masses loaded. (B) Cytoplasmic and nuclear fractions of HEK293 control and CD63 CRISPR cells transfected with GFP or GFP-LMP1 were separated and confirmed by enrichment of cytoplasmic calnexin or nuclear histone H4 protein, equal masses loaded. (C) Akt and ERK activation in cytoplasmic fractions of HEK293 and CD63 CRISPR cells following GFP-LMP1 transfection was measured, equal masses loaded. Relative levels of phospho-Akt and phospho-ERK were averaged over three independent experiments. (D) HA-LMP1 packaging in EVs (equal volumes) from Rat1 cells stably expressing a pBabe-HA-LMP1 vector following CD63 knockout. (E) Quantitation of HA-LMP1 packaging in Rat1 EVs from three independent experiments. (F) Focus formation assay was performed using Rat1 control or CD63 knockout cells transduced with an empty pBabe vector or pBabe-HA-LMP1. (G and H) Immunoblot analysis of LMP1-induced NF-κB signaling activation in cytoplasmic (G) and nuclear (H) fractions of HEK293 control and CD63 CRISPR cells, equal masses loaded. The results shown are representative of findings from multiple experiments.

Article Snippet: Blots were probed with primary antibodies against the following: Alix (Q-19; Santa Cruz Biotechnology), HSC70 (B-6; Santa Cruz), TSG101 (C-2; Santa Cruz), calnexin (11397; Santa Cruz), caveolin-1 (D46G3; Cell Signaling), flotillin-2 (H-90; Santa Cruz), CD63 (TS63; Abcam), GFP (600-101-215; Rockland), HA (C29F4; Cell Signaling), LMP1 (CS1-4; Dako), histone H4 ( 81 , 82 ), IKKα (11930; Cell Signaling), IKKβ (8943; Cell Signaling), phospho-IKKα/β (2697; Cell Signaling), IκBα (4814; Cell Signaling), phospho-IκBα (2859; Cell Signaling), NF-κB p65 (8242; Cell Signaling), phospho-NF-κB p65 (3033; Cell Signaling), RelB (4922; Cell Signaling), NF-κB p100/p52 (3017; Cell Signaling), phospho-NF-κB p100/p52 (4810; Cell Signaling), ERK 2 (C-14; Santa Cruz), phospho-p44/42 MAPK (9106; Cell Signaling), Akt (9272; Cell Signaling), phospho-Akt (4060; Cell Signaling), and phospho-STAT3 (9134; Cell Signaling).

Techniques: Knock-Out, Transfection, Isolation, Western Blot, CRISPR, Activation Assay, Stable Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Tube Formation Assay, Transduction

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet: In the mid-exponential phase ( t = 3.5 hr), E. coli MG1655 wild-type and knockout strains underwent ( A ) streptomycin, ( B ) gentamicin, and ( C ) amikacin treatments at a concentration of 50 μg/ml for a duration of 5 hr in test tubes. Following the treatments, cells were washed to eliminate the antibiotics and then plated on LB agar plates to quantify the surviving cell fractions. CFU: colony-forming units; WT: wild type. For pairwise comparisons, one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used where **p < 0.01, ***p < 0.001, and ****p < 0.0001. N = 3. Data points represent mean and standard error.

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Knock-Out, Concentration Assay

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet: ( A ) Growth of E. coli MG1655 wild type, Δ sucA , Δ gltA , Δ nuoI , and Δ icd strains was assessed by measuring the number of cells per ml with flow cytometry. ( B ) The cells of both the E. coli MG1655 WT and mutant strains were collected from culture flasks (see Materials and methods for details) at indicated time intervals and then subjected to gentamicin treatment. The figure shows the colony-forming unit (CFU) levels of both the treated and untreated cultures, indicating the surviving and total cell populations, respectively. N = 3. Data points represent mean and standard error.

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Flow Cytometry, Mutagenesis

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet: In the mid-exponential phase ( t = 3.5 hr), E. coli MG1655 wild-type and knockout strains underwent gentamicin treatments at a concentration of 5 μg/ml for a duration of 5 hr in test tubes. Following the treatments, cells were washed to eliminate the antibiotics and then plated on LB agar plates to quantify the surviving cell fractions. For pairwise comparisons, one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used where ***p < 0.001 and ****p < 0.0001. N = 4. Data points represent mean and standard error.

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Knock-Out, Concentration Assay

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet: E. coli MG1655 wild-type cells in the exponential phase ( t = 3.5 hr) were subjected to staining with 1 μM redox sensor green (RSG) for 10 min at 37°C, followed by analysis using flow cytometry. Controls included unstained cells and cells treated with a combination of 20 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and 1 μM RSG. CCCP was introduced into cell suspensions 5 min before RSG staining, aiming to reduce cellular redox activities. The presented panel serves as a representative biological replicate.

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Staining, Flow Cytometry

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet: The pH vs. fluorescence ratio (410/470 nm) was established for E. coli MG1655. The standard curve data were fitted using a Boltzmann sigmoid curve.

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Fluorescence

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet: ( A ) During the mid-exponential phase ( t = 3.5 hr), ATP synthase knockout strains of E. coli BW25113 were subjected to gentamicin treatment at a concentration of 50 μg/ml in test tubes for 5 hr. After the treatments, cells were washed to remove the antibiotics and then plated on LB agar plates to quantify the surviving cell fractions in terms of colony-forming units (CFU). ( B ) The cells of E. coli MG1655 wild-type and ATP synthase knockout strains were collected from flasks at indicated time intervals and then subjected to gentamicin treatment. The figure shows CFU levels of both the treated and untreated cultures. For pairwise comparisons, a one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was employed, where ****p < 0.0001. N = 3. Data points represent mean and standard error.

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Knock-Out, Concentration Assay

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet: The cells of E. coli MG1655 wild-type and ATP synthase knockout strains were exposed to 5 μg/ml gentamicin at the mid-exponential phase ( t = 3.5 hr) for 5 hr in test tubes. After the treatments, cells were washed to remove the antibiotics and then plated on LB agar plates to quantify CFU levels. For pairwise comparisons, one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used where ****p < 0.0001. N = 4. Data points represent mean and standard error.

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Knock-Out

Journal: eLife

Article Title: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

doi: 10.7554/eLife.94903

Figure Lengend Snippet:

Article Snippet: The UniProt-SwissProt E. coli K12 database (Taxon ID 83333, downloaded on 6/19/2023, 4518 entries) served as the reference.

Techniques: Mutagenesis, Recombinant, Viability Assay, Software