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