Journal: bioRxiv

Article Title: Cargo-selective regulation of clathrin-mediated endocytosis by AMP-activated protein kinase

doi: 10.1101/2024.11.05.622177

Figure Lengend Snippet: (A) Arf6-WT-eGFP-RPE cells stimulated with 1 μM doxycycline for 24 hours were treated with 100 μM A- 769662 or vehicle control for 5 minutes, fixed, labelled with an anti-clathrin antibody, and imaged by TIRF-M. Scale bar represents 5 μm. (B) Automated detection and analysis of Arf6-WT-eGFP intensity in clathrin structures is shown as the mean ± SD from four independent experiments. Control: k (cells) = 33 and n (CLSs) = 5137; A-769662: k (cells) = 44 and n (CLSs) = 6466. * p<0.05 [Mann-Whitney U test]. The data presented here is a part of the experiment that is also shown in and Figure S6A . (C) Arf6-WT-UltraID cells stimulated with 1 μM doxycycline (dox) for 24 hours were treated with 50 μM biotin in the presence of 100 μM A-769662 or vehicle control for 15 minutes. Shown is a representative immunoblot of whole cell lysates (input) and biotinylated interactors isolated by streptavidin pulldown (IP) detected by western blotting using the indicated antibodies. (D) Relative abundance of AP2 normalized to Arf6-WT-UltraID expression shown as the mean ± SD from six independent experiments. * p<0.05 [Wilcoxon signed-rank test]. (E) eGFP-CLCa-RPE cells transfected with Arf6 siRNA or non-targeting control siRNA were treated with 100 μM A-769662 or vehicle control for 5 minutes. Shown are TIRF-M images of fixed cells labelled with an anti-Dab2 antibody. Scale bar represents 5 μm. (F) Automated detection and analysis of Dab2 intensity in clathrin structures is shown as a representative experiment ( left ) and as the mean ± SD from five independent experiments ( right ). Control siRNA, control: k (cells) = 177 and n (CLSs) = 74005; control siRNA, A-769662: k (cells) = 166 and n (CLSs) = 76139; Arf6 siRNA, control: k (cells) = 184 and n (CLSs) = 89747; Arf6 siRNA, A-769662: k (cells) = 190 and n (CLSs) = 91736. * p<0.05 [two-way ANOVA with a Šídák post-hoc test]. The data presented here is a part of the experiment that is also shown in and Figure S7C . (G) ARPE-19 cells transfected with Arf6 siRNA or non-targeting control siRNA were treated with 100 μM A-769662 or vehicle control for 5 minutes and antibody labelled for cell surface β1-integrin (antibody clone 4B7). Scale bar represents 20 μm. (H) Mean fluorescence intensity of cell surface β1-integrin is shown as a representative experiment ( left ) and as the mean ± SD from four independent experiments ( right ) that analyzed 30-50 individual cells per condition. * p<0.05 [two-way ANOVA with a Fisher’s LSD test].

Article Snippet: Cell lysates were plated onto enzyme-linked immunosorbent assay (ELISA) plates coated with either anti-Tfn (Bethyl Laboratories) or anti-EGF (Millipore Sigma) antibodies, followed by detection of biotin-xx-Tfn or biotin-xx-EGF (free, unbound to avidin) by incubation with streptavidin conjugated to horseradish peroxidase (POD; BioBasic), and detection of bound POD by o-phenylenediamine assay.

Techniques: Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Transfection, Fluorescence

Journal: Frontiers in Immunology

Article Title: Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs

doi: 10.3389/fimmu.2022.827760

Figure Lengend Snippet: Regulation of cytokines in primary human PBMCs during infection with HK and its HA variants. (A) mRNA expression of cytokines ( IFN-α, CCL5, IL-6 ) and nucleic acid sensor ( RIG-I ) of PBMCs inoculated at MOI 3 with HK and its HA variants HK-H17R and HK-R2 measured 6 hours p.i. using RT-PCR. Mean expression values +SD of four donors in duplicates are shown. Raw data of cytokines were normalized to the expression of the housekeeping gene RPS11 and related to HK infected samples. Significance determined by ANOVA followed by Turkey’s multiple comparison test. P values shown if significant *p < 0.05, **p < 0.01, ***p < 0.001. (B, C) MACS-isolated pDCs were either inoculated with recombinant IAVs at MOI 1 or stimulated with synthetic TLR-ligand ODN 2216 for 24 hours. Concentrations of (B) IFN-α or (C) IL-6 in the cell supernatants were determined by ELISA and depicted as mean amount of cytokine [ng/ml] +SD of two donors in duplicates.

Article Snippet: The following antibodies were used: capture: rat anti-human IL-6 (Pharmingen, 554543) or anti-human IFN-α coating antibody (Bender, 2010-10), detection: biotin rat anti-human IL-6 (Pharmingen, 554546) followed by streptavidin-POD conjugate (Roche, 11089153001) or anti-human-IFN-α HRP-conjugate (eBioscience, BM216MSTK).

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs

doi: 10.3389/fimmu.2022.827760

Figure Lengend Snippet: Regulation of cytokines in primary human PBMCs during infection with HK and its HA variants. (A) mRNA expression of cytokines ( IFN-α, CCL5, IL-6 ) and nucleic acid sensor ( RIG-I ) of PBMCs inoculated at MOI 3 with HK and its HA variants HK-H17R and HK-R2 measured 6 hours p.i. using RT-PCR. Mean expression values +SD of four donors in duplicates are shown. Raw data of cytokines were normalized to the expression of the housekeeping gene RPS11 and related to HK infected samples. Significance determined by ANOVA followed by Turkey’s multiple comparison test. P values shown if significant *p < 0.05, **p < 0.01, ***p < 0.001. (B, C) MACS-isolated pDCs were either inoculated with recombinant IAVs at MOI 1 or stimulated with synthetic TLR-ligand ODN 2216 for 24 hours. Concentrations of (B) IFN-α or (C) IL-6 in the cell supernatants were determined by ELISA and depicted as mean amount of cytokine [ng/ml] +SD of two donors in duplicates.

Article Snippet: The following antibodies were used: capture: rat anti-human IL-6 (Pharmingen, 554543) or anti-human IFN-α coating antibody (Bender, 2010-10), detection: biotin rat anti-human IL-6 (Pharmingen, 554546) followed by streptavidin-POD conjugate (Roche, 11089153001) or anti-human-IFN-α HRP-conjugate (eBioscience, BM216MSTK).

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs

doi: 10.3389/fimmu.2022.827760

Figure Lengend Snippet: Regulation of cytokines in primary human PBMCs during infection with HK and its HA variants. (A) mRNA expression of cytokines ( IFN-α, CCL5, IL-6 ) and nucleic acid sensor ( RIG-I ) of PBMCs inoculated at MOI 3 with HK and its HA variants HK-H17R and HK-R2 measured 6 hours p.i. using RT-PCR. Mean expression values +SD of four donors in duplicates are shown. Raw data of cytokines were normalized to the expression of the housekeeping gene RPS11 and related to HK infected samples. Significance determined by ANOVA followed by Turkey’s multiple comparison test. P values shown if significant *p < 0.05, **p < 0.01, ***p < 0.001. (B, C) MACS-isolated pDCs were either inoculated with recombinant IAVs at MOI 1 or stimulated with synthetic TLR-ligand ODN 2216 for 24 hours. Concentrations of (B) IFN-α or (C) IL-6 in the cell supernatants were determined by ELISA and depicted as mean amount of cytokine [ng/ml] +SD of two donors in duplicates.

Article Snippet: The following antibodies were used: capture: rat anti-human IL-6 (Pharmingen, 554543) or anti-human IFN-α coating antibody (Bender, 2010-10), detection: biotin rat anti-human IL-6 (Pharmingen, 554546) followed by streptavidin-POD conjugate (Roche, 11089153001) or anti-human-IFN-α HRP-conjugate (eBioscience, BM216MSTK).

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: IL-18 Induces Airway Hyperresponsiveness and Pulmonary Inflammation via CD4 + T Cell and IL-13

doi: 10.1371/journal.pone.0054623

Figure Lengend Snippet: (A) The concentrations of IFN-γ, IL-13, IL-1β, and eotaxin in the bronchoalveolar lavage fluids (BALFs) were measured by specific ELISA kits. (n = 4 to 6 per each group) *: p<0.05 (B) On day 19, BALF cells were isolated from OVA-sensitized and challenged mice. Three-color analysis was performed for intracellular cytokine staining. Isolated BALF cells were stained with FITC-anti-mouse CD4 mAb, PE-anti-mouse IL-13 mAb, PC7-anti-mouse IFN-γ mAb, and/or control isotype matched mAbs, as reported previously . The lymphocyte population was gated for intracellular cytokine analysis. (C) Mice were anesthetized intraperitoneally with sodium pentobarbital, and their tracheas were cannulated via tracheostomy. The mice were ventilated mechanically (tidal volume, 325 µl; frequency, 120 breaths/minute). A paralytic agent (pancuronium bromide) was administered and airway opening pressure was measured with a differential pressure transducer and was recorded continuously. Stepwise increases of acetylcholine chloride (ACh, catalog no. A-6625, Sigma–Aldrich Chemical) in 0.9% saline (0.6 to 160 mg/ml) were given by an ultrasonic nebulizer (30 seconds). The data were expressed as the provocative concentration 200 (PC200); the concentration at which airway pressure was 200% of its baseline. PC200 was calculated by log-linear interpolation for individual mice. The data were also expressed as airway resistance changes from baseline in response to 9 different doses of Ach (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 mg/ml). (n = 6 to 10 per each group) *: p<0.05. (D) Serum Total IgE and OVA-specific IgE concentrations were measured. (n = 5 to 12 per each group) *: p<0.05.

Article Snippet: Plates were washed and incubated with 3 μg of biotin-labeled OVA in PBS, washed, incubated with streptavidin-horseradish peroxidase conjugate, washed, and developed with ELISA POD substrate TMB kit (Nacalai tesque, Kyoto, Japan).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Staining, Control, Saline, Concentration Assay