Journal: bioRxiv

Article Title: Toll-like receptor signalling via IRAK4 confers epithelial integrity and tightness through regulation of junctional tension

doi: 10.1101/2021.10.25.465823

Figure Lengend Snippet: Localisation of TLR1 ( A-A’’’ ), TLR2 ( B-B’’’ ) and the downstream scaffold protein MyD88 ( C-C’’’ ) in epithelial Caco-2 cells. Z-projections of apical-most confocal sections are shown. Antibody labelling against TLR1, 2 and MyD88 is in green, phalloidin and ZO-1 in magenta are used to label cell boundaries. A’’ , B’’ and C’’ show an apical-basal cross-section of the epithelium (nuclei stained with DAPI in blue), apical is up. A’’’ , B’’’ and C’’’ show magnifications of the channels for TLR1, 2 or MyD88, respectively, the insets show the overlay. D TLR1, TLR2, TLR4 and TLR 6 are all detected in confluent Caco-2 cells during maturation over 3 weeks by Western Blotting. Note that TLR1 levels consistently drop in fully matured cells. E Levels of phospho-IRAK4 (pIRAK4) increase with maturation of Caco-2 cells in culture, indicating steady-state pathway activation and increased signalling with more mature junctions. F In innate immunity signalling, TLR binding of an immuno-active ligand triggers assembly of the Myddosome containing pIRAK4, IRAK1 and MyD88, leading to IκB phosphorylation, dissociation from NFκB that is now free to enter the nucleus and affect transcription. A constitutive, non-immune role of the TLR pathway would not require a ligand but might go through the same pathway. See also .

Article Snippet: Fluorescent TLR1, TLR2, TLR4, TLR6, and Myd88 constructs (13014, 13015, 13016, 13018,13020) were obtained from Addgene.

Techniques: Staining, Western Blot, Activation Assay, Binding Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Toll-like receptor signalling via IRAK4 confers epithelial integrity and tightness through regulation of junctional tension

doi: 10.1101/2021.10.25.465823

Figure Lengend Snippet: A, B Western blot analysis of protein levels in 1-week post-confluence Caco-2 monolayers upon treatment with siRNAs targeting TLR2 (1-4) or TLR4 as control ( A ) as well as TLR1 ( B ), to demonstrate a reduction in protein level for TLR2 and TLR1. C siRNA treatment of Caco-2 monolayers with different siRNAs targeting TLR1, TLR2 or IRAK4 compared to a scramble control leads to reduction in TEER level. n= 5 independent transwells (scramble, siTLR1, siTLR2, siIRAK4) were averaged. Statistical significance was determined by one way ANOVA with Dunnett’s multiple comparison test as * = p<0.01, ** = p<0.001, **** = p<0.00001. D-O In siRNA-treated 1-week post-confluence Caco-2 monolayers with reduced TLR1 or TLR2 levels, localisation of junctional components to cell borders is affected. Z-projections of confocal sections covering the apical-lateral junctional area are shown. D-F Knock-down of TLR1and TLR2 leads to ZO-1 reduction at junctions of 44.5% and 30.0%, respectively. G-I Knock-down of TLR1and TLR2 leads to mild Occludin reduction at junctions of 14.3% and 9.7%, respectively. J-L Knock-down of TLR1and TLR2 leads to reduction of Claudin-3 at junctions of 27.3% and 49.2%, respectively. M-O Knock-down of TLR1and TLR2 leads to reduction of Claudin-5 at junctions of 41.2% and 30.3%, respectively. P-S Quantification of components in scramble control, TLR1 siRNA and TLR2 siRNA treated Caco-2 cells. P ZO-1: n= 267 junctions were analysed for the Scramble control and n= 160 for siTLR1 and n=216 for siTLR2. Q Occludin: n= 111 junctions were analysed for the scramble control and n= 125 for siTLR1 and n=93 for siTLR2. R Claudin-3: n= 141 junctions were analysed for the scramble control and n= 175 for siTLR1 and n=142 for siTLR2. S Claudin-5: n= 202 junctions were analysed for the scramble control and n= 168 for siTLR1 and n=187 for siTLR2. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparison test as** = p<0.001, **** = p<0.00001. Note that scramble values for Claudin-5 and Occludin are the same as those in as these were run contemporaneously. T Analysis of phospho-α-Catenin levels upon treatment with siRNA against TLR2 (1-4) or against TLR4 compared to scramble control. GAPDH is shown as loading control. U Analysis of phospho-α-Catenin levels upon treatment with siRNA against TLR1, TLR5, MyD88 orIRAK1 compared to scramble control. GAPDH is shown as loading control. V Model of ‘constitutive’ TLR pathway function compared to the immune-response-induced pathway. See also .

Article Snippet: Fluorescent TLR1, TLR2, TLR4, TLR6, and Myd88 constructs (13014, 13015, 13016, 13018,13020) were obtained from Addgene.

Techniques: Western Blot, Control, Comparison, Knockdown

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: Colocalization of N-cadherin and SEPT7 during the endocytosis of C. albicans . Endothelial cells were infected with wild-type C. albicans for the indicated times and then fixed and stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields (HPF) that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7 were counted at the indicated times after infection. Results are the means ± standard deviations (SD) from three experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e, i) and SEPT7 (b, f, j) accumulating around the same organism. The merged images are shown in panels c, g, and k, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d, h, and l. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Infection, Staining

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: SEPT7 and actin colocalize during the endocytosis of C. albicans . Endothelial cells were infected with C. albicans for the indicated times and then fixed and stained for SEPT7 and actin. (A) The numbers of organisms in 15 HPF that were surrounded by SEPT7 alone, actin alone, or SEPT7 plus actin were counted at the indicated times after infection. Results are the means ± SD from three experiments, each performed in triplicate. (B) Confocal microscopic images of SEPT7 (a, e, i) and actin (b, f, j) accumulation. The merged images are shown in panels c, g, and k, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d, h, and l. Arrows indicate the SEPT7 and actin that accumulated around the hyphae. Scale bar = 5 µm.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Infection, Staining

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: C. albicans binds to a complex of N-cadherin and SEPT7. Endothelial cells were infected with C. albicans for the indicated time points, and the endothelial cell proteins that bound to the organisms, either directly or indirectly (bound), were isolated. The nonbinding proteins (total) were also collected. The bound and total N-cadherin and SEPT7 were identified by immunoblotting. (A) Representative immunoblot. (B) Densitometric analysis of 3 independent immunoblots comparing the relative amounts of bound to total protein. Results are means ± SD. (C) Endothelial cells were infected with C. albicans for 90 min and lysed, after which the lysates were subjected to immunoprecipitation (IP) with either control IgG or an anti-N-cadherin antibody. The presence of SEPT7 and N-cadherin in immunoprecipitated proteins was detected by immunoblotting.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Infection, Isolation, Western Blot, Immunoprecipitation, Control

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: Effects of N-cadherin siRNA on C. albicans recruitment of endothelial cell N-cadherin and SEPT7. Endothelial cells were transfected with either control or N-cadherin siRNA and then infected with wild-type C. albicans for 90 min. (A, B) The cells were fixed and then stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7. Results are the means ± SD from three experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e) and SEPT7 (b, f) accumulating around the same organism. The merged images are shown in panels c and g, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d and h. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm. (C) Representative immunoblot showing the effects of N-cadherin knockdown on the amount of endothelial cell N-cadherin and SEPT7 that bound to C. albicans hyphae. (D) Densitometric analysis of 3 independent SEPT7 immunoblots. Results are means ± SD. *, P ≤ 0.05 compared to cells transfected with control siRNA.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Transfection, Control, Infection, Staining, Western Blot, Knockdown

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: Als3 and Ssa1 mediate binding to the N-cadherin–SEPT7 complex. Endothelial cells were infected for 90 min with the indicated strains of C. albicans or with S. cerevisiae containing the backbone vector (pADH1 or pYES2.1) or expressing C. albicans ALS3 (pALS3) or SSA1 (pSSA1). Next, the endothelial cell proteins that bound to these strains were isolated and then identified by immunoblotting.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Binding Assay, Infection, Plasmid Preparation, Expressing, Isolation, Western Blot

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: SEPT7 knockdown reduces the accumulation of N-cadherin around C. albicans . Endothelial cells were transfected with either control or SEPT7 siRNA and then infected with C. albicans for 90 min. (A, B) The cells were fixed and then stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7. Results are the means ± SD from three experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e) and SEPT7 (b, f) accumulating around the same organism. The merged images are shown in panels c and g, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d and h. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm. (C) Representative immunoblot showing the effects of SEPT7 knockdown on the amount of endothelial cell N-cadherin and SEPT7 that bound to C. albicans hyphae. (D) Densitometric analysis of 4 independent N-cadherin immunoblots. Results are means ± SD. *, P < 0.05 compared to cells transfected with control siRNA.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Knockdown, Transfection, Control, Infection, Staining, Western Blot

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: Cytochalasin D inhibits the interaction of N-cadherin and SEPT7 with C. albicans . Endothelial cells were incubated with diluent alone (control) or cytochalasin D (cyto. D) and then infected with C. albicans for 90 min. (A, B) The cells were fixed and then stained for N-cadherin and SEPT7. (A) The number of organisms in 15 high-power fields that were surrounded by N-cadherin alone, SEPT7 alone, or N-cadherin plus SEPT7. Results are the means ± SD from five experiments, each performed in triplicate. (B) Confocal microscopic images of N-cadherin (a, e) and SEPT7 (b, f) accumulating around the same organism. The merged images are shown in panels c and g, and images of the corresponding microscopic fields viewed by differential interference contrast are shown in panels d and h. Arrows indicate the N-cadherin and SEPT7 that accumulated around the hyphae. Scale bar = 5 µm. (C) Representative immunoblot showing the effects of cytochalasin D on the amount of endothelial cell N-cadherin and SEPT7 that bound to C. albicans hyphae. (D) Densitometric analysis of 5 independent N-cadherin and SEPT7 immunoblots. Results are means ± SD. *, P < 0.05 compared to control cells; † , P < 0.05 compared to N-cadherin. (E, F) Coimmunoprecipitation results showing the effects of cytochalasin D on the association between SEPT7 and N-cadherin. (E) Representative immunoblot of cell lysates that were immunoprecipitated with either an anti-N-cadherin antibody or control IgG, separated by SDS-PAGE and then probed for the presence of SEPT7 by immunoblotting. (F) Densitometric analysis of 3 independent immunoblots. Results are means ± SD. *, P = 0.03 compared to control cells that had been infected with C. albicans in the absence of cytochalasin D.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Incubation, Control, Infection, Staining, Western Blot, Immunoprecipitation, SDS Page

Journal: mBio

Article Title: Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

doi: 10.1128/mBio.00542-13

Figure Lengend Snippet: SEPT7 knockdown inhibits endocytosis of C. albicans . (A) Endothelial cells transfected with either control or N-cadherin siRNA were infected with C. albicans for 90 min, after which the number of endocytosed organisms was determined by a differential fluorescence assay. Results are the means ± SD from three experiments, each performed in triplicate. *, P < 0.001 compared to endothelial cells transfected with the control siRNA. (B) Verification by immunoblotting of SEPT7 knockdown, which did not affect total N-cadherin content.

Article Snippet: Cells were then rinsed with Hank’s balanced salt solution (HBSS), fixed with 3% paraformaldehyde in PBS, and then blocked and permeabilized with 0.05% Triton X-100 in 5% goat serum for 30 min. SEPT7 was labeled with rabbit anti-SEPT7 polyclonal antibody (catalog no. 13018-1-AP; Protein Tech Group, Inc.).

Techniques: Knockdown, Transfection, Control, Infection, Fluorescence, Western Blot