Fibronectin

For photoactivation experiments, HT1080 WT or ACBD3 stably knockdown cells were seeded on a glass-bottom dish (35 mm diameter, Cellvis) coated with fibronectin (Millipore). After 18 h co-transfection with KDELR1-PA-GFP and indicated mCherry-Rab, cells were imaged with a 63× objective on a Zeiss LSM 880 confocal microscope in an atmosphere of 5% CO₂ at 37 °C. Photoactivating of KDELR1-PA-GFP in the Golgi was achieved using a 405 nm laser. Images were acquired every 5 s for 5 min.
For live cell imaging, HeLa WT or ACBD3-KO cells were seeded on a glass-bottom dish (35 mm diameter, Cellvis) coated with fibronectin (Millipore). After 18 h co-transfection with GT-GFP and mCherry-Rab4A, cells were imaged with a 63× objective on a Zeiss LSM 880 ariyscan confocal microscope in an atmosphere of 5% CO₂ at 37 °C. Images were acquired every 5 s for 5 min.

To subject hCF to global strain, we used custom-made stretchable culture membranes that were functionalized with 2 μg/cm² fibronectin (Millipore) [39 (link)]. HCF were cultured at 1000 cells per cm² for 24 h before straining the cultures by 20% in a custom-built uniaxial stretching device for 1 h. To strain hCF at sites of FAs, we used magnetic microparticles (3 µm diameter, Fe₃O₄, Sigma, Oakville, ON, Canada), coated with 100 μg/mL fibronectin (Millipore) [35 (link),41 (link)]. fibronectin-coated microbeads were added to hCF cultures for 30 min, followed by three washes with medium to remove unbound microbeads. A ceramic permanent magnet was then applied to generate tensile forces perpendicular to the cells’ growth plane via the attached microbeads for 1 h. Microbead-bound proteins were isolated by washing cells with PBS and scraping into extraction buffer (0.5% Triton X-100, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM 1,4-Piperazinediethanesulfonic acid, pH 6.8), supplemented with protease inhibitors cocktail (Sigma). The suspension was sonicated and the microbead fraction was separated from the cytosolic fraction using the magnetic separation stand. Bead fractions were suspended in ice-cold extraction buffer, homogenized, and boiled in sample buffer for 5 min. Microbeads were pelleted and supernatant was collected for Western blotting.

A similar protocol to that described above was used to produce an N-hydroxysuccinimide (NHS) ester-functionalized ICC scaffold, with a pre-polymer solution containing 50% (w/v) PEG-DA, 10% (w/v) acryloyl-PEG-NHS (Laysan Bio, AL), and 0.05% photo initiator in deionized water. The prepared PEG-NHS ICC scaffold was coated with either collagen I or fibronectin by centrifugation, shaking, and incubation in 20 μg/ml collagen I (Merck Millipore, Germany) or fibronectin (Merck Millipore) solution in pH 7.4 PBS at 4 °C overnight. Excess collagen I or fibronectin was removed by repeated washing with PBS.
To characterize the ECM coating, the ICC scaffolds were fixed in 4% paraformaldehyde (Alfa Aesar, MA) then incubated with a mouse primary antibody against collagen I or fibronectin (Abcam, UK) at 4 °C overnight in the presence of 3% bovine serum albumin (BSA; Sigma-Aldrich) and labeled with an anti-mouse secondary antibody conjugated to a fluorophore. Green fluorescent Alexa Fluor 488 was used to label collagen I and red fluorescent Alexa Fluor 555 was used to label fibronectin. After PBS washing, the ECM-functionalized scaffolds were imaged using a Carl Zeiss LSM 710 confocal microscope.

Single donor human neonatal dermal lymphatic microvascular endothelial cells (ndLECs) were obtained from Lonza (Visp, Switzerland, catalogue #CC-2812; HMVEC-dLyNeo) and cultured in complete EBM-2 medium consisting of EBM-2 basal Medium (CC-3156, Lonza) supplemented with EGM-2 MV Single Quots^TM (CC-4147, Lonza). Cell culture dishes were pre-coated with 10μg/ml collagen type 1 (Advanced Biomatrix) and 10μg/ml fibronectin (Merck Millipore) in PBS.
Juvenile skin LECs (jdLECs) from the foreskin of a single male donor (Lot Nr. 431Z006.2) were obtained from Promocell (Heidelberg, Germany, # C-12216) and cultured in complete EBM-2 (CC-3156, Lonza) supplemented with EGM^TM-2 SingleQuots^TM (CC-4176, Lonza), without VEGF-A (CC-4176, Lonza), additionally supplemented with (3%) FBS for a final concentration of 5% FBS (Gibco), as used for ndLECs. Cell culture dishes were pre-coated with 10μg/ml collagen type 1 (Advanced Biomatrix, San Diego, CA, USA) and 10μg/ml fibronectin (Merck Millipore) in PBS.

Prior to embryo dissection, tissue cultureware (Celltreat, 229168-chambered microscope slides were used for imaging) were coated with 25 ug/ml fibronectin (Sigma, F1141) and incubated for at least 45 min in a 37 °C humidified incubator maintained at 5% CO₂ atmosphere. After incubation, the fibronectin solution was removed from the cultureware and DMEM (Sigma, D6046) supplemented with 10% FBS (Thermo Fisher Scientific, 10-437-028) was added. NCC explants were obtained by thinly micro-dissecting dorsal neural folds from HH9 (6–7 somite) embryos in Ringer’s solution as previously described^{18 (link)}. Each dissected neural fold was then aspirated in a minimal amount of Ringer’s solution (2.5 ul) and placed in a well of the fibronectin-coated tissue cultureware containing media. The explants were incubated for at least 12 h under normoxic conditions in a humidified 37 °C incubator maintained at 5% CO₂ atmosphere. The duration of incubation was determined by the appropriate conditions pertaining to specific experiments.