cd49e 160gd  (fluidigm)

Journal: Nature microbiology

Article Title: Aspergillus fumigatus CalA Binds to Integrin α5β1 and Mediates Host Cell Invasion

doi: 10.1038/nmicrobiol.2016.211

Figure Lengend Snippet: a, Immunoblot of biotin-labeled A549 epithelial cell membrane proteins that had been eluted from hyphae of the indicated strains of A fumigatus. Blot was probed with an anti-biotin antibody. Arrow indicates the band containing β1 integrin. Results are representative of 3 independent experiments. b, Immunoblot of membrane proteins from A549 epithelial (epith) cells (top) and vascular endothelial (endo) cells (bottom) that had been eluted from the indicated A. fumigatus strains. Blots were probed with an antibody against β1 integrin (Total memb, total membrane). c, Densitometric analysis of 5 immunoblots from A549 cells and 3 immunoblots from endothelial cells as in (b). *P < 0.04 vs. Af293 and the ΔcalA+calA complemented strain (two-tailed Student's t-test assuming unequal variances). d-f, Effects of antibodies against integrin β1 (int β1) and α5 (int α5) on the endocytosis of wild-type A. fumigatus by A549 pulmonary epithelial cells (d) primary vascular endothelial cells (e), and primary human alveolar epithelial cells (f). Results are the mean ± SD of three experiments, each performed in triplicate. *P < 0.001 vs. control IgG (two-tailed Student's t-test assuming unequal variances). HPF, high-powered field. g-h, Effects of siRNA knockdown of integrin β1 and α5 on the endocytosis of wild-type A. fumigatus by A549 pulmonary epithelial cells (g) and primary vascular endothelial cells (h). Results are the mean ± SD of three experiments, each performed in triplicate. *P < 0.005 vs. control siRNA (two-tailed Student's t-test assuming unequal variances). i, Immunoblots of whole cell lysates showing siRNA knockdown of integrin β1 and α5. Results are representative of 3 independent experiments. j-k, Endocytosis of the indicated strains of A. fumigatus (j) and S. cerevisiae expressing ALS1 alone (ALS1) or ALS1 and calA (ALS1+calA) (k) by the GD25 (integrin β1 deficient) and β1AGD25 (integrin β1 restored) cell lines. Results are the mean ± SD of three experiments, each performed in triplicate. *P < 0.005 vs. the same strain on GD25 cells, † p < 0.01 vs. A. fumigatus Af293 or Als1-expressing S. cerevisiae on the same host cell line (two-tailed Student's t -test assuming unequal variances).

Article Snippet: Antibody blocking, siRNA, and inhibitors For antibody blocking experiments, host cells were incubated with 10 μg/mL of the anti-β1 integrin antibody (6S6, Millipore), 25 μg/mL of the anti-α5 integrin antibody (NKI-SAM-1, Millipore), or 25 μg/ml control mouse IgG for 45 min before infection.

Techniques: Western Blot, Labeling, Membrane, Two Tailed Test, Control, Knockdown, Expressing

Journal: Nature microbiology

Article Title: Aspergillus fumigatus CalA Binds to Integrin α5β1 and Mediates Host Cell Invasion

doi: 10.1038/nmicrobiol.2016.211

Figure Lengend Snippet: a, Immunoblots of cell membrane proteins from A549 epithelial cells (top) and vascular endothelial cells (bottom) that had been eluted from the indicated strains of A. fumigatus. Blots were probed with an antibody against integrin α5. b, Densitometric analysis of 4 immunoblots of samples from A549 cells and 3 immunoblots of samples from endothelial cells, such as the ones in (a). *P < 0.04 vs. Af293 and the ΔcalA+calA complemented strain (two-tailed Student's t-test assuming unequal variances). c, Confocal microscopic images showing the accumulation of integrin α5β1 integrin around A. fumigatus during infection of A549 epithelial cells and vascular endothelial cells. Results are representative of 3 independent experiments. Thin arrows indicate the accumulation of integrin around the organisms. Hollow arrows indicate the organisms that are shown at higher magnification in the insets. Scale bar, 20 μm. d,e, Effects of the indicated peptides (50 μm) on the endocytosis of A fumigatus by epithelial cells (d) and endothelial cells (e). Results are the mean ± SD of three experiments, each performed in triplicate. *P < 0.01 vs. control or cells incubated with the RGD peptide (two-tailed Student's t-test assuming unequal variances). HPF, high-powered field. f, Images of S. cerevisiae cells expressing either CalA and Als1 or Als1 alone that were incubated with recombinant integrin α5β1 and the stained with an anti-integrin α5 antibody. Results are representative of 3 independent experiments. Scale bar, 5 μm.

Article Snippet: Antibody blocking, siRNA, and inhibitors For antibody blocking experiments, host cells were incubated with 10 μg/mL of the anti-β1 integrin antibody (6S6, Millipore), 25 μg/mL of the anti-α5 integrin antibody (NKI-SAM-1, Millipore), or 25 μg/ml control mouse IgG for 45 min before infection.

Techniques: Western Blot, Membrane, Two Tailed Test, Infection, Control, Incubation, Expressing, Recombinant, Staining