Journal: Journal of Visualized Experiments : JoVE

Article Title: Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

doi: 10.3791/3791

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Article Snippet: L17 , Biotinylated Solanum Tuberosum (Potato) Lectin , STL , Vector Laboratories , BK-3000.

Techniques: Plasmid Preparation, Positive Control

Journal: Biochimica et biophysica acta. Biomembranes

Article Title: The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast

doi: 10.1016/j.bbamem.2020.183255

Figure Lengend Snippet: Ctr_1, Ctr_2, Ctr_3 and HsAFP1_1, HsAFP1_2, HsAFP1_3 represent the control and HsAFP1 treatment of 3 independent experiments, respectively. C. albicans SC5314 cells were treated for 150 minutes with HsAFP1 (2x FC50; 10 μg/mL) in PDB/YPD with 50 mM HEPES pH 7. Colored bars indicate the Z-score, representing the relative expression of a gene compared to the mean of expression for the corresponding gene over all six samples. Up- and down-regulated genes coding for: GPI-anchored proteins, GPI-anchor synthesis, GPI-anchor building block biosynthesis and phosphatidylinositol-specific phospholipase C (PI-specific PLC). Down-regulated iron- and copper-related genes and up-regulation of calcium-related genes. Up- and down-regulated genes involved in macro- or mitophagy, respectively. Up-regulated genes coding for: cell wall biosynthesis and cell cycle enzymes as well as cellular bud located proteins.

Article Snippet: Yeast cells were cultured in the following liquid media: YPD (yeast extract (10 g/L; LabM, UK), peptone (20 g/L; LabM, UK) and glucose (20 g/L; Sigma-Aldrich, USA), YNB (yeast nitrogen base without amino acids; MP Biomedicals, USA) (6.7 g/L), PDB/YPD (potato dextrose broth (19.2 g/L; BD, USA), yeast extract (2 g/L), peptone (4 g/L) and glucose (4 g/L)) adjusted to pH 7 with 50 mM HEPES (Sigma-Aldrich, USA), synthetic complete (SC) medium (CSM (complete amino acid supplement mixture; MP Biomedicals, USA) (0.77 g/L), YNB (6.7 g/L) and glucose (20 g/L)) adjusted to pH 7 with 50 mM HEPES or 1/5 th PDB/YNB; at 30°C and 37°C for S. cerevisiae and C. albicans , respectively.

Techniques: Expressing, Blocking Assay

Journal: Biochimica et biophysica acta. Biomembranes

Article Title: The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast

doi: 10.1016/j.bbamem.2020.183255

Figure Lengend Snippet: S. cerevisiae WT and Δbst1 mutant cells were treated for 150 minutes in PDB/YPD with 50 mM HEPES pH 7 at 30°C. BODIPY-HsAFP1 and propidium Iodide (PI; 3 μM) were used as markers for peptide internalization and cell death, respectively. (A) Confocal microscope images of S. cerevisiae cells treated with high HsAFP1 doses (10x FC50; 250 μg/mL). Scale bar: 5 μm. (B) Flow cytometry of WT and Δbst1 mutant cells. WT cells are co-administrated with BODIPY-HsAFP1 (1/2 FC50; 12.5 μg/mL, FC50; 25 μg/mL and 2x FC50; 50 μg/mL)and 2 mM CaSO4, 2 mM CuSO4, 2 mM MgSO4 or 2 mM FeSO4. The % of cells that do have BODIPY-HsAFP1 associated with their surface or internalized (green) and the % of cells that do not have BODIPY-HsAFP1 associated with their surface or internalized (grey) is presented. (C) Flow cytometry of WT and Δbst1 mutant cells. WT cells are co-administrated with BODIPY-HsAFP1 (1/2 FC50; 12.5 μg/mL, FC50; 25 μg/mL and 2x FC50; 50 μg/mL) and 2 mM CaSO4, 2 mM CuSO4, 2 mM MgSO4 or 2 mM FeSO4. The % of cells that both have BODIPY-HsAFP1 associated with their surface/internalized and have compromised membranes is presented in red. All other subpopulations are presented in grey, including the % of cells that do not have BODIPY-HsAFP1 internalized or associated with their surface and do not have compromised membranes, the % of cells that do not have BODIPY-HsAFP1 internalized or associated with their surface and do have compromised membranes, and the % of cells that do have BODIPY-HsAFP1 internalized or associated with their surface and do not have compromised membranes. Data are means ± SD for n ≥ 3 independent experiments. To analyze significant differences in the size of the subpopulations, two-way ANOVA followed by Tukey multiple comparison was performed, with *** and **** representing P < 0.001 and P < 0.0001, respectively.

Article Snippet: Yeast cells were cultured in the following liquid media: YPD (yeast extract (10 g/L; LabM, UK), peptone (20 g/L; LabM, UK) and glucose (20 g/L; Sigma-Aldrich, USA), YNB (yeast nitrogen base without amino acids; MP Biomedicals, USA) (6.7 g/L), PDB/YPD (potato dextrose broth (19.2 g/L; BD, USA), yeast extract (2 g/L), peptone (4 g/L) and glucose (4 g/L)) adjusted to pH 7 with 50 mM HEPES (Sigma-Aldrich, USA), synthetic complete (SC) medium (CSM (complete amino acid supplement mixture; MP Biomedicals, USA) (0.77 g/L), YNB (6.7 g/L) and glucose (20 g/L)) adjusted to pH 7 with 50 mM HEPES or 1/5 th PDB/YNB; at 30°C and 37°C for S. cerevisiae and C. albicans , respectively.

Techniques: Mutagenesis, Microscopy, Flow Cytometry

Journal: Biochimica et biophysica acta. Biomembranes

Article Title: The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast

doi: 10.1016/j.bbamem.2020.183255

Figure Lengend Snippet: (A) S. cerevisiae WT cells treated 150 min with HsAFP1 (1/2 FC50; 12.5 μg/mL, FC50; 25 μg/mL and 2x FC50; 50 μg/mL) or HsAFP1[H32A][R52A] doses (equivalent to the highest tested HsAFP1 dose) in PDB/YPD with 50 mM HEPES pH 7 at 30°C. Dose-dependent increase of the vacuolar pH and decrease of survival of HsAFP1-treated S. cerevisiae cells as compared to the control (MQ water; black bars), determined via the vacuolar dye BCECF-AM and CFU counting, respectively. Concanamycin A (ConA) was used as positive control for increased vacuolar pH, whereas cell survival was not affected. Equimolar HsAFP1[H32A][R52A] doses (as the highest tested HsAFP1 dose) did not affect vacuolar pH or survival. Means ± SD are presented for n ≥ 3 independent experiments. Significant differences in vacuolar pH or Log10 numbers of CFU between the negative control (MQ water; black bars) and all other treatments were determined via one-way ANOVA followed by Dunnett multiple comparison, with **, *** and **** representing, P < 0.01, P < 0.001 and P < 0.0001, respectively (presented by orange bars). (B) Decrease in replicative lifespan of S. cerevisiae BY4742 cells by HsAFP1 treatment under dietary restriction (DR) conditions, as represented by the dotted arrow. Replicative lifespan of cells on agar plates in the presence or absence of HsAFP1 and containing 2% or 0.05% glucose (= dextrose (D)), with 0.05% glucose representing DR conditions. The HsAFP1 dose used is the maximum dose that does not affect replicative lifespan in 2% glucose. Replicative lifespan results are presented from an experiment containing n = 40 cells tested per condition.

Article Snippet: Yeast cells were cultured in the following liquid media: YPD (yeast extract (10 g/L; LabM, UK), peptone (20 g/L; LabM, UK) and glucose (20 g/L; Sigma-Aldrich, USA), YNB (yeast nitrogen base without amino acids; MP Biomedicals, USA) (6.7 g/L), PDB/YPD (potato dextrose broth (19.2 g/L; BD, USA), yeast extract (2 g/L), peptone (4 g/L) and glucose (4 g/L)) adjusted to pH 7 with 50 mM HEPES (Sigma-Aldrich, USA), synthetic complete (SC) medium (CSM (complete amino acid supplement mixture; MP Biomedicals, USA) (0.77 g/L), YNB (6.7 g/L) and glucose (20 g/L)) adjusted to pH 7 with 50 mM HEPES or 1/5 th PDB/YNB; at 30°C and 37°C for S. cerevisiae and C. albicans , respectively.

Techniques: Positive Control, Negative Control