Fibronectin f2006

Mouse anti-LAMP1 monoclonal antibody (555798) was purchased from BD Biosciences; mouse anti-diphosphorylated ERK1/2 monoclonal antibody (M8159) was from Sigma-Aldrich; rabbit anti-ERK1/2 polyclonal antibody (V114A) was from Promega; mouse anti-β-actin monoclonal antibody (G043) was from Abm; mouse anti-γ-tubulin antibody (T6557) was from Sigma-Aldrich; goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) (sc-2031) and goat anti-rabbit IgG-HRP polyclonal antibody (sc-3837) were from Santa Cruz Biotechnology; goat anti-mouse IgG-TRITC antibody (T5393) was from Sigma-Aldrich; BSA (A7906) was from Sigma-Aldrich; SDS-PAGE reagents were from Bio-Rad; fetal bovine serum (FBS) was from Gibco; LysoTracker (L7528) was from Thermo Fisher Scientific; fibronectin (F2006) was from Sigma-Aldrich; FGF2 was from PeproTech; and Alcian blue dye (74240) was from EuroDiagnostica. The recombinant NK1 fragment of HGF was produced using yeast Pichia pastoris expression system and purified with heparin affinity chromatography as previously described.³⁹ (link)

For live-cell imaging, 35-mm glass-bottom dishes (BS-20-GJM, Biosharp, China) were coated with 10 μg/mL fibronectin (F2006; Sigma, MO, USA) in PBS for at least 3 h at 37°C, washed with PBS twice, and immersed in complete DMEM without phenol red (PM150211; Procell, China) before seeding of cells. The time-lapse images of cells with transient transfection of hABHD16A-GFP and hIFITM1-DsRed were acquired with Leica TCS SP8 laser scanning confocal microscope. Appropriate filters, a heated sample environment (37°C), controlled 5% CO₂, and a ×60/1.5 oil objective were used. The recording was set as every 10 s for 360 s, and one focal plane was recorded for all live cell videos.

The migration of cells under different chemokine gradients was analyzed using ibiTreat 2D “mu (µ)‐Slide” Chemotaxis system (Ibidi GmbH, Martinsried, Germany).^[28] Before the chemotaxis assay, the surface of the slide for was coated with fibronectin (F2006, 300 µg mL⁻¹, Sigma) for 1 h for cells attachment. After washing with PBS, 6 µL of cells (3 × 10⁶ cells mL⁻¹) were loaded into the central observation channel and incubated at 37 °C for 45 min to allow attachment. Gradients of CXCL12 were generated according to the manufacturer's instructions. For the migration track analysis, live cell imaging was recorded for indicated periods and intervals on a JuLi stage system (NanoEnTek, Seoul, Korea) operating on a CO₂ incubator. The tracking of migrating individual cells was achieved using the Manual Tracking plugin (Institut Curie, Orsay, France) in ImageJ software (NIH, Bethesda, MA). Chemotaxis plots were obtained with the Chemotaxis and Migration plugin from Ibidi. To analyze the actin dynamics of live cells during chemotaxis, the cells were stained with SiR‐Actin, a fluorogenic, cell permeable and highly specific probe for F‐actin (SC001, 1 × 10⁻⁶m, Spirochrome, Stein am Rhein, Switzerland) for 1 h before seeding onto “mu‐Slide” and time‐lapse images were obtained using confocal microscope (Nikon) with a 60× oil objective and a temperature‐controlled chamber (37 °C, 5% CO₂).