Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: (A and B) Hyphae of the indicated strains of C. albicans were incubated for 45 min with human umbilical vein endothelial cells (A) or the FaDu oral epithelial cell line (B), after which the number of endocytosed and cell-associated (endocytosed plus adherent) organisms was determined by a differential fluorescence assay. The data are expressed as a percentage of the results with wild-type organisms, and are the mean ± SD of three experiments, each performed in triplicate. *, p < 0.001 compared to the wild-type strain and the als3 Δ/ als3 Δ:: ALS3 -complemented strain. (C and D) Binding of surface proteins from endothelial cells (C) and FaDu oral epithelial cells (D) to hyphae of the indicated strains of C. albicans . Biotinylated endothelial or epithelial cell membrane proteins that bound to C. albicans were eluted from the hyphae with urea and separated by SDS-PAGE. (C) The same immunoblot of endothelial cell surface proteins was developed with an anti-biotin monoclonal antibody (top) and an anti-N-cadherin monoclonal antibody (bottom). (D) An immunoblot of oral epithelial cell surface proteins was developed with an anti-biotin monoclonal antibody (top) and an anti-E-cadherin monoclonal antibody (bottom). Arrows in (C) and (D) indicate the bands that correspond to N-cadherin and E-cadherin, respectively.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Incubation, Fluorescence, Binding Assay, Membrane, SDS Page, Western Blot

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: (A) Number of hyphae of the indicated clinical isolates of C. albicans that were endocytosed by and cell-associated with the FaDu oral epithelial cell line after a 45-min incubation. The data are expressed as a percentage of the results with strain DAY185, and are the mean ± SD of three experiments, each performed in triplicate. *, p < 0.05 compared to strain DAY185. (B and C) Immunoblot of biotinylated FaDu cell surface proteins eluted from the indicated strains of C. albicans. The same blot was probed with antibodies against biotin (B) or E-cadherin (C). (D) Flow cytometric detection of Als3 expression on the surface of C. albicans hyphae. Hyphae of the indicated strains were stained using indirect immunofluorescence with either a polyclonal rabbit anti-Als3 antiserum (solid lines) or purified rabbit IgG (broken lines) and then analyzed using flow cytometry.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Incubation, Western Blot, Expressing, Staining, Immunofluorescence, Purification, Flow Cytometry

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: Confocal micrographs of uninfected endothelial cells (A–C), or endothelial cells infected with the wild-type strain (D–G), the als3 Δ/ als3 Δ null mutant (H–K), or the als3 Δ/ als3 Δ:: ALS3 -complemented strain (L–O). The cells were stained for N-cadherin (A), (D), (H), and (L), actin microfilaments (B), (E), (I), and (M), and C. albicans (F), (J), and (N). The merged images are shown in (C), (G), (K), and (O). Arrows indicate the accumulation of N-cadherin and microfilaments around the organisms. Bar in (O) indicates 10 μm.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Infection, Mutagenesis, Staining

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: Confocal micrographs of uninfected FaDu oral epithelial cells (A–C) or epithelial cells infected with the wild-type strain (D–G), the als3 Δ/ als3 Δ null mutant (H–K), or the als3 Δ/ als3 Δ:: ALS3 -complemented strain (L–O). The cells were stained for E-cadherin (A), (D), (H), and (L), microfilaments (B), (E), (I), and (M), and C. albicans (F), (J), and (N). The merged images are shown in (C), (G), (K), and (O). Arrows indicate the accumulation of E-cadherin and microfilaments around the organisms. Bar in (O) indicates 10 μm.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Infection, Mutagenesis, Staining

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: (A–D) Latex beads were coated with BSA, rAls1-N, or rAls3-N. They were incubated for 45 min with endothelial cells (A), FaDu oral epithelial cells (B), CHO cells expressing human N-cadherin (C), or CHO cells expressing human E-cadherin (D), after which the number of endocytosed and cell-associated beads was determined. (E) The interactions of BSA- and rAls3-N–coated beads with control CHO cells expressing no human cadherins (Control) were compared with those of CHO cells expressing human E-cadherin (E-cadherin) by the same method as in (A–D). Data are expressed as a percentage of the results with beads coated with BSA and are the mean ± SD of three or four experiments, each performed in triplicate. *, p ≤ 0.005 compared to beads coated with BSA; † p < 0.05 compared to beads coated with BSA; § p < 0.005 compared to beads coated with rAls1-N; and ‡ p ≤ 0.01 compared to control CHO cells.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Incubation, Expressing, Control

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: The most distal (N1) and adjacent (N2) N-terminal domains of Als1, Als3, E-cadherin, and N-cadherin are shown. The predicted structures of the N-terminal regions of Als1 and Als3 were extracted from their larger models, and structures for E-cadherin and N-cadherin were obtained from the Protein Data Bank . (A) Comparative secondary structures. Yellow color indicates β-sheet propensity; red, α-helical propensity; aqua, extended or turn structures. Note that the Als1 model encompasses 187 amino acids (residues 3–190) in an equivalent 3-D space as that of Als3, which includes 279 amino acids (residues 20–299). Such a comparison illustrates the overall similarities in conformation among these polypeptide domains, with a more compact configuration of Als3. (B) Comparative hydrophobic surfaces. Surface hydrophobicity is illustrated in color upon the solvent-accessible surface areas of respective molecules. For clarity, only the hydrophobic surface is colored; the ghost structures (gray) represent the polar surface. In hydrophobic areas, brown indicates the most hydrophobic regions and blue indicates the least hydrophobic regions. (C) Comparative electrostatic surfaces. Negatively charged electrostatic surface is illustrated in color upon the solvent accessible surface areas of respective molecules. For clarity, only the negatively charged surface is colored; the ghost structures (gray) represent the small area on each molecule bearing a positive charge. In negatively charged areas blue indicates the strongest negative surface potential and yellow indicates the weakest negative charge potential.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Comparison, Solvent

Journal: PLoS Biology

Article Title: Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

doi: 10.1371/journal.pbio.0050064

Figure Lengend Snippet: The interactions of the N-terminal regions of Als1 and Als3 with the N-terminal regions of N-cadherin and E-cadherin, as well as N-cadherin and E-cadherin self-association were modeled as outlined in Materials and Methods. The N-terminal domains of Als1 and Als3 are shown in their energy-minimized conformations; docking results using alternate conformations yielded equivalent results (unpublished data). (A) The solvent-accessible surface areas of the proteins are shown, and the β barrel domains are numbered sequentially from the N-terminus. In models of the docking of an Als protein to a cadherin (left and middle columns), the Als protein is blue and the cadherin is gold. In models of cadherin self-association, one cadherin molecule is blue and the other cadherin is gold. (B) Interactions of Als1 or Als3 with E-cadherin or N-cadherin are presented to illustrate their predicted docking characteristics. In the top two rows, the indicated cadherin electrostatic contact surfaces are superimposed upon the backbone structures of Als1 or Als3. Coloration schema of the Als secondary structure: gold, β sheet; blue, extended/turn. Coloration schema of the cadherin surface: blue, most negative; red, most positive. In the bottom two rows, the contact footprints of the indicated cadherins (dark blue) are superimposed upon the solvent-accessible surface areas of Als1 or Als3 (aqua) to indicate the predicted docking positions of the protein pairs. In all cases, note that the ectodomain N2 of Als3 contributes to the interactions with both cadherins, while the corresponding ectodomain N2 of Als1 does not participate in interactions with either cadherin.

Article Snippet: The eluted proteins were separated by SDS-PAGE on an 8% gel and detected by immunoblotting with an anti-biotin murine monoclonal antibody (clone BN-32; Sigma-Aldrich), an anti-N-cadherin murine monoclonal antibody (clone 32; Transduction Laboratories, http://www.bdbiosciences.com ), or the anti-E-cadherin murine monoclonal antibody.

Techniques: Solvent

Journal: Frontiers in Pharmacology

Article Title: Berberine Protects Against NLRP3 Inflammasome via Ameliorating Autophagic Impairment in MPTP-Induced Parkinson’s Disease Model

doi: 10.3389/fphar.2020.618787

Figure Lengend Snippet: BBR mitigates autophagic impairment in MPTP-induced mice. (A) The representative immunohistochemical staining for MAP1LC3B in SN. (B) The number of MAP1LC3B positive cells in SN. Representative western blot bands (C) and the statistical graph (D) of MAP1LC3B and BECN1 in SN. (E) Transmission electron microscopy shown with autophagosomes (yellow arrows) in the SN. Data were expressed as the mean ± SD ( n = 6). * p < 0.05 compared with control group, # p < 0.05 compared with MPTP group, + p < 0.05 compared with MPTP + BBR group. MAP1LC3B, microtubule associated protein 1 light chain 3 beta; SN, substantia nigra; BECN1, beclin 1.

Article Snippet: The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026).

Techniques: Immunohistochemical staining, Staining, Western Blot, Transmission Assay, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Berberine Protects Against NLRP3 Inflammasome via Ameliorating Autophagic Impairment in MPTP-Induced Parkinson’s Disease Model

doi: 10.3389/fphar.2020.618787

Figure Lengend Snippet: BBR enhances autophagic activity in MPP + -treated BV2 cells. The representative immunofluorescent staining (A) and puncta (B) of MAP1LC3B in BV2 cells treated with MPP + at 200 μM and BBR at the concentration of 12.5, 25, and 50 μM (20 cells were analyzed per group for MAP1LC3B puncta counting). Representative western blots (C) and the statistical graph (D) of MAP1LC3B and BECN1 in BV2 cells treated with MPP + at 200 μM and BBR at the concentration of 12.5, 25, and 50 μM. The representative double-immunofluorescent staining (E) and puncta (E) of MAP1LC3B in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM (20 cells were analyzed per group for MAP1LC3B puncta counting). The representative monodansylcadaverine staining (G) and statistical graph (H) of autophagic vesicles in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM. Representative western blot bands (I) and the statistical graph (J) of MAP1LC3B and BECN1 in BV2 cells treated with MPP + at 200 μM, BBR at 25 μM and 3-MA at 10 mM. Data were expressed as the mean ± SD ( n = 3). * p < 0.05 compared with untreated group, # p < 0.05 compared with MPP + group, + p < 0.05 compared with MPP + + BBR group. MAP1LC3B, microtubule associated protein 1 light chain 3 beta; BECN1, beclin 1.

Article Snippet: The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026).

Techniques: Activity Assay, Staining, Concentration Assay, Western Blot