Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Engineered Biomimetic Fibrillar Fibronectin Matrices Regulate Cell Adhesion Initiation, Migration, and Proliferation via α5β1 Integrin and Syndecan-4 Crosstalk.

doi: 10.1002/advs.202300812

Figure Lengend Snippet: Figure 3. Syndecan-4 crosstalks with 𝛼5𝛽1 integrin to sense the fibrillarity and mechanical stiffness of FN. a,b) Adhesion forces of pKO-𝛽1 fibroblasts to heparin-treated 2D globular FN substrates, 2.5D fibrillar FN matrices, and 3D fibrillar FN matrices a) without and b) with crosslinking. Reference adhesion forces of pKO-𝛽1 fibroblasts to non-treated FN substrates or matrices are given in gray. Bottom p values compare given and reference data. Middle p values compare 2.5D fibrillar or 3D fibrillar FN matrices with 2D globular FN substrates. Top p values compare 2.5D fibrillar and 3D fibrillar FN matrices. AS indicates the slope of a linear regression and s.e. of adhesion strengthening rate (pN s−1). c) Adhesion forces of pKO-𝛽1 fibroblasts depleted from syndecan-1 (SDC1 KO), syndecan-2 (SDC2 KO), syndecan-3 (SDC3 KO), or syndecan-4 (SDC4 KO) to 2D globular, 3D fibrillar, and crosslinked 3D fibrillar (3DX) FN substrates. Adhesion forces of pKO-𝛽1 fibroblasts to respective FN substrates are given in gray as reference. p values compare given and reference data. d) Adhesion forces of pKO-𝛽1 SDC4 KO fibroblasts rescued with syndecan-4 to 2D globular, 3D fibrillar, and crosslinked 3D fibrillar (3DX) FN substrates. Adhesion forces of pKO-𝛽1 fibroblasts to respective FN substrates are given in gray as reference. Bottom p values compare given and reference data. Middle p values compare 2.5D fibrillar or 3D fibrillar FN matrices with 2D globular FN substrates. Top p values compare 2.5D fibrillar and 3D fibrillar FN matrices. Dots represent adhesion forces of individual fibroblasts and red bars the median. n indicates the number of fibroblasts tested. p values were calculated using two-tailed Mann–Whitney test.

Article Snippet: The detached fibroblasts were resuspended in SCFS media with FBS (1% w/v) and recovered from detachment process at 37 °C for at least 30 min. After recovery, cells were pelleted and 106 fibroblasts were resuspended in flow cytometry buffer (100 μL, EDTA (2 mm) and BSA (2% w/v) in PBS) containing antibodies as follows; phycoerythrin (PE)-labeled integrin subunit β1 (1:6, BioLegend, 102 208), SDC1 (1:10, R&D systems, FAB2966P) with rat IgG control (1:10, R&D systems, IC005P), or SDC4 (1:50, Abcam, ab279590) with rabbit IgG control (1:50, Abcam, ab209478) antibodies/Alexa Fluor 647-conjugated SDC2 (1:20, R&D systems, FAB6585R) with sheep IgG control (1:20, R&D systems, IC016R) antibodies at 4 °C for 1 h. For SDC3, 106 fibroblasts were incubated with SDC 3 (1:400, ThermoFisher, PA5-47377) with Goat IgG control (1:400, ThermoFisher, 02–6202) antibodies in flow cytometry buffer at 4 °C for 1 h, washed by flow cytometry media twice, and incubated with Alexa Fluor 488 rabbit anti-goat IgG (Invitrogen, A11078) at 4 °C for 1 h. After incubation, fibroblasts were washed by flow cytometry buffer twice, followed by measuring their fluorescence intensity employing LSRFortessa (BD AG).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pathological Allergen-Specific B Cells by Protein-Engineered Molecules in a Mouse Model of Chronic House Dust Mite Allergy

doi: 10.3390/ijms252413661

Figure Lengend Snippet: Phenotyping of immune cells in the lungs by FACS analyses. ( A ) Characterization of myeloid cell types—alveolar macrophages ( I ), SiglecF high CD11c low ( III ), SiglecF med CD11c− ( IV ) and total eosinophils ( II ), and neutrophils ( V )—presented as the percentage of the parent population. ( B ) Analysis of B and antibody-secreting cells—percentages of CD19 cells ( I ), CD19 CD80 cells ( II ), CD19 IgE cells ( III ), CD138 cells ( IV ), plasma cells ( V ), plasmablasts ( VI ), and CD32 mean fluorescent intensity (MFI) of CD19 ( VII ) and CD19 IgE cells ( VIII ). ( C ) Phenotyping of T cells—CD3 cells ( I ), CD3 CD4 cells ( II ), CD3 CD8 cells ( III ), CD4 CD69 − CD25 + cells ( IV ), CD4 CD69 + CD25 + cells ( V ), CD4 CD69 + CD25 − cells ( VI ), CD8 CD69 − CD25 + cells ( VII ), CD8 CD69 + CD25 − cells ( VIII ), and CD4 CD69 + CD25 + cells ( IX ). Data are shown as mean ± SD of 8–9 mice per group. The differences between the groups were evaluated using a one-way ANOVA followed by Tukey’s multiple comparisons, or a Kruskal–Wallis test followed by Dunn’s multiple comparisons test, depending on the normality of the data; p values are indicated on the graphs: * p < 0.05; ** p < 0.01; *** p < 0.001. Data are representative of at least 4 independent experiments.

Article Snippet: The following were used for the fluorescence-activated cell sorting (FACS) experiments: Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD16/32 antibody (Cat# 35-0161-U100, clone 2.4G2, Tonbo Bioscience, San Diego, CA, USA), anti-rat IgG-FITC antibody (Cat# 405404, BioLegend, San Diego, CA, USA), anti-mouse FITC-conjugated CD11c (Cat# 117306, clone N418, BioLegend, San Diego, CA, USA), CD25 (Cat# 102006, clone PC61, BioLegend, San Diego, CA, USA), and CD45R (B220) (Cat# 103206, clone RA3-6B2, BioLegend, San Diego, CA, USA); Phycoerythrin (PE)-conjugated CD19 (Cat# 115508, clone 6D5, BioLegend, San Diego, CA, USA), SiglecF (Cat# 12-1702-80, clone 1RNM44N, eBioscience, Frankfurt, Germany), CD69 (Cat# 104508, clone H1.2F3, BioLegend, San Diego, CA, USA), and CD138 (Cat# FAB2966P, clone 300506, R&D Systems, Minneapolis, MN, USA); PE-Cyanine 7 (PE-Cy7)–conjugated CD3 (Cat# 100320, clone 145-2C11, BioLegend, San Diego, CA, USA) and CD8 (Cat# 100722, clone 53-6.7, BioLegend, San Diego, CA, USA); Pacific Blue-conjugated Ly6G (Cat# 127612, clone 1A8, BioLegend, San Diego, CA, USA), CD3 (Cat# 100334, clone 145-2C11, BioLegend, San Diego, CA, USA), and CD80 (Cat# 104724, clone 16-10A1, BioLegend, San Diego, CA, USA); PE-Cyanine 5 (PE-Cy5)–conjugated CD4 (Cat# 100410, clone GK1.5, BioLegend, San Diego, CA, USA) and Streptavidin (Cat# 15-4317-82, eBioscience, San Diego, CA, USA); and biotin-conjugated IgE (Cat# 13-5992-85, clone 23G3, eBioscience, San Diego, CA, USA) mAbs.

Techniques: Clinical Proteomics