Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Vibrio vulnificus RtxA1 Toxin Expression Upon Contact With Host Cells Is RpoS-Dependent

doi: 10.3389/fcimb.2018.00070

Figure Lengend Snippet: V. vulnificus- induced cytotoxicity and morphological changes in HeLa cells were decreased by an rpoS mutation. (A) HeLa cells in 48-well plates were infected with either V. vulnificus wild-type (wt), an rpoS mutant ( rpoS − ), or an rtxA1 mutant ( rtxA1 − ) strains at an MOI of 20. Lactate dehydrogenase released into the culture supernatant was assayed as a marker of cytotoxicity. (B) HeLa cells in 24-well plates were infected with V. vulnificus strains at an MOI 20 for 120 min. The bacterial suspension 10-fold serially diluted (10 μL) were loaded on HI agar plates overnight and the viable bacterial cells were counted. (C) HeLa cells cultured in an 8-well-chambered coverslip were infected with V. vulnificus strains at an MOI of 100 for 75 min. The cytoplasmic membranes were stained with Alexa Fluor 594-conjugated WGA (red color). (D) Restoration of the rpoS gene expression in the rpoS complemented strain ( rpoS − +pLAFR3:: rpoS ) was determined by conventional RT-PCR. The 16S rRNA housekeeping gene was used as an internal control. The fragments were resolved by electrophoresis on 1% agarose gel. (E) HeLa cells were infected with V. vulnificus strains with vector pLAFR3 at an MOI of 20 for 180 min, and the cytotoxicity was measured by LDH assay. Results represent the average of at least three independent experiments. Values are means ± SD (vs. V. vulnificus wild-type, ** P

Article Snippet: The HeLa cells were then incubated with Alexa Fluor 594-conjugated wheat germ agglutinin (WGA, red color) (ThermoFisher Scientific) for 10 min to visualize the cytoplasmic membranes.

Techniques: Mutagenesis, Infection, Marker, Cell Culture, Staining, Whole Genome Amplification, Expressing, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Lactate Dehydrogenase Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass

doi: 10.1073/pnas.1511285112

Figure Lengend Snippet: ( A ) Representative bright field images of TRAP-stained OCs grown by coculture of WT and Tmem178 −/− BMMs and BMSC as indicated. ( B ) Representative bright field images of resorptive pits in WT and Tmem178 −/− BMMs cultured on bovine bone slices in the presence of 10 ng/mL M-CSF and 50 ng/mL RANKL for 10 d. Resorption pits were visualized by staining with peroxidase-conjugated wheat-germ agglutinin. Black lines delineate the resorbed areas. n = 8–9 per genotype. ( C ) Quantification of resorbed area in images from B . * P

Article Snippet: Cells were then removed from the bone surface by using sodium hydroxide and gentle agitation, and bone slices were stained with 20 µg/mL peroxidase-conjugated wheat-germ agglutinin (Sigma) for 30 min at room temperature, followed by incubation with 3,3′-diaminobenzidine (0.52 mg/mL in PBS containing 0.1% H2 O2 ) for 30 min.

Techniques: Staining, Cell Culture

Journal: Scientific Reports

Article Title: Proteomic profile of extracellular vesicles released by Lactiplantibacillus plantarum BGAN8 and their internalization by non-polarized HT29 cell line

doi: 10.1038/s41598-020-78920-z

Figure Lengend Snippet: Inhibitors of clathrin-mediated endocytosis block internalization of L. plantarum EVs in HT29 cells. ( A ) The left panel shows the vesicle uptake in the absence of endocytosis inhibitors: HT29 cells incubated with rhodamine B-R18-labeled EVs (1 μg/well) from L. plantarum (red circles); controls: EVs (grey squares) and HT29 cells (brown triangles). The right panels show the uptake of L. plantarum EVs in the presence of inhibitors of endocytosis pathways: HT29 cells pre-incubated (prior to the addition of rhodamine B-R18-labeled EVs) with the lipid raft disrupting agents filipin III (blue diamonds) or nystatin (green triangles), or with the clathrin-mediated endocytosis (CME) inhibitors chlorpromazine (yellow triangles) or dynasore (purple diamonds). The uptake in the absence of inhibitors is shown as a control (red circles). Fluorescence intensity was normalized by fluorescence detected at the indicated time points by labelled EVs in the absence of HT29 cells. Data are presented as means ± standard error from three independent experiments. Statistical differences were assessed using one-way ANOVA followed by Tukey’s test: *, significance against untreated control cells ( p ≤ 0.001); **, significance against uptake values in the absence of inhibitors ( p ≤ 0.012). ( B ) Visualization of internalized EVs by confocal fluorescence microscopy in the absence (no inhibitor) and the presence of the inhibitors dynasore and nystatin after 1 h and 4 h of incubation. Control cells: HT29 in the absence of EVs. Nuclei were stained with DAPI (blue), the cell membrane with WGA-Alexa Fluor-488 (green) and internalized EVs with rhodamine B-R18-label (red). Scale bar: 20 μm.

Article Snippet: After 1 h and 4 h, cells were washed with PBS and the nuclei were labeled with DAPI whereas membranes were labeled with Alexa Fluor-488 wheat germ agglutinin (WGA, Molecular Probes) (1 μg/ml, 4 °C), after incubation for 25 min, followed by fixation for 30 min with 3% paraformaldehyde.

Techniques: Blocking Assay, Incubation, Labeling, Fluorescence, Microscopy, Staining, Whole Genome Amplification