Tumour necrosis factor α

Apical versus basolateral sEV production by PTEC was examined using Transwell^® plates (Corning, Cambridge, MA, USA). Human primary PTEC were seeded onto 6‐well transparent polyester Transwell inserts (0.4 μm pore size, 24 mm diameter, 4.67 cm² surface area) at a concentration of 1.2 × 10⁵ cells/cm² in DM (2.5 ml volume in the upper compartment). DM alone (3.1 ml) was added to the lower compartment. PTEC were grown to confluence as confirmed in permeability studies described below.
Once monolayer integrity was confirmed, the DM in the upper and lower compartments was exchanged with fresh DM for normal control PTEC and fresh DM supplemented with 100 ng/ml interferon (IFN)‐γ and 20 ng/ml tumour necrosis factor (TNF)‐α (both from R&D Systems, Minneapolis, MN, USA) for inflammatory PTEC and then further cultured for 72 h. PTEC culture medium was subsequently harvested from the upper (apical) compartment (2.5 ml for each Transwell) and lower (basolateral) compartment (3.1 ml for each Transwell). Individual collections of apical and basolateral media for each culture condition from each PTEC donor were pooled for downstream sEV isolation.

Cell culture media and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, US) unless stated otherwise. A23178, naproxen, celecoxib calcipotriol hydrate, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. Nordihydroguaiaretic acid (NDGA) was from Cayman chemicals (Ann Arbor, MI, USA) Recombinant human epidermal growth factor (EGF) and tumour necrosis factor (TNF)-α were from R&D systems (Abdingdon, UK). The fluoroketone AVX001 was synthesized and characterized according to Holmeide and Skattebol [39 (link)], and provided by Dr. Inger Reidun Aukrust and Dr. Marcel Sandberg (Synthetica AS, Oslo, Norway). AVX001 was stored at −80 °C as a 20 mM stock solution in dimethyl sulphoxide (DMSO) under argon gas to minimize oxidation.

A keratinocyte growth medium (KGM) was purchased from Kurabo Industries, Ltd. (Osaka, Japan). The nylon membranes were obtained from Millipore (Bedford, MA, USA). Recombinant human interleukin (IL)‐4, IL‐5, tumour necrosis factor (TNF)‐α, macrophage migration inhibitory factor (MIF), and IL‐1β were ordered from R&D Systems (Minneapolis, MN, USA). The antiserine racemase (ab123894) monoclonal antibody was from Abcam (Cambridge, MA, USA), and anti‐β‐actin antibody was from Sigma‐Aldrich Co (St. Louis, MO, USA). The detection system for Western blot was purchased from Cell Signaling Technology (Beverly, MA, USA). The other reagents used in the study were of analytical grade.

Rabbit anti-lumican (cat.ab168348) and E-cadherin (cat.ab40772) monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-Slug (cat.9585), β-actin (cat.4970), alpha-smooth muscle actin (α-SMA; cat.19245), p44/42 MAPK (ERK; cat.4695), and phospho-p44/42 MAPK (p-ERK; cat.4370) monoclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-alpha-1 type III collagen (COL3A1; cat.A3795) polyclonal antibody was purchased from ABclonal Technology (Wuhan, China). Mouse anti-fibronectin monoclonal antibodies (cat.250073) was purchased from Zen Bioscience (Chengdu, China). Alexa Fluor® 488 goat anti-rabbit (cat.4412) and Alexa Fluor® 594 goat anti-mouse (cat.8890) secondary antibodies were purchased from Cell Signaling Technology. Recombinant human interleukin (IL)-6, IL-8, tumour necrosis factor (TNF)-α, and lumican proteins were purchased from R&D Systems (Minneapolis, MN, USA).