0.4 μm transwell insert

hMSCs (p4-p10) were seeded in 75 cm² flask and cultured to 80% confluence. After washed with PBS for three times, serum-free medium with or without 10 ng/ml TNF-α was added. After 24 h, medium without TNF-α pretreatment was collected, filtered, and stored at −80 °C as CM. Alternatively, cells with TNF-α pretreatment were washed with PBS for three times, and cultured for another 24 h in fresh serum-free medium. After 24 h, medium was collected, filtered, and stored at −80 °C as TCM. For coculture experiment, 0.4 μm Transwell insert (Corning, New York, USA) was applied. MSCs cultured in serum-free media with or without TNF-α pretreatment were seeded in the upper chamber, while tumor cells were seeded in the lower chamber. As the control for CM or TCM treatment, serum-free α-MEM was used for culturing colon cancer cell lines.

For coculture experiments, OBs were plated onto 6-well plates (20×10³ cells/well). Twenty-four hours later, MKs (20×10³ cells/well) were plated into wells for direct coculture. For indirect coculture, OBs were seeded in the upper chamber of a 0.4 μm transwell insert (Corning, New York, USA) and MKs were seeded in the lower wells. Specified cultures were pretreated with 100 nM of SB431542 (Sigma-Aldrich, St. Louis, Missouri, USA) for OBs for approximately 1 h. MKs conditioned medium (MKs-CM) was obtained by removing MKs via centrifugation (5000 rpm, 10 min). In some assays, neutralizing antibodies against TGF-β1 (9016; R&D, Minneapolis, Minnesota, USA ), VEGF (AF-493; R&D), BMP6 (MAB6325; R&D), IGF-1 (AF791; R&D), PDGF-BB (ab34074; Abcam), CXCl12 (79014; R&D), BMP2 (MAB111; R&D), TGF-β2 (AB-112-NA; R&D), TGF-β3 (20724; R&D), BMP4 (MAB50201; R&D) and IgG (AB-108-C; R&D) were added to the MKs-CM.

Cancer cells were cultured in the lower chamber in the presence or absence of trastuzumab and PBMCs were added to the upper chamber using the 0.4 μm transwell insert (Corning). After 48 h co-culturing, PBMCs were removed from top chamber and cancer cells were prepared for WB analysis.

This study was approved by the National Healthcare Group Domain-Specific Review Board of Singapore (Ethics approval number: DSRB D/11/228; IRB 13–509). Nasal epithelial biopsies were obtained from adult patients with inferior turbinate or nasal polyps, who were scheduled for septoplasty or polypectomy in the Department of Otolaryngology of the National University Hospital (Singapore). These fresh nasal epithelial tissues were used to derive human nasal epithelial stem/progenitor cells (hNESPCs) and then differentiated into human nasal epithelial cells (hNECs) in air-liquid interface (ALI) culture system as described previously [13 (link), 16 (link), 17 (link)]. Briefly, primary cells were expanded with B-ALI™ complete growth medium (Lonza, Walkersville, MD) for about 1 week, and then transferred onto 12-well 0.4 μm Transwell inserts (Corning, Corning, NY, USA). 4 days after seeding, growth medium was discarded and 700 μl of PneumaCult™-ALI Medium with inducer supplements (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) was added to the basal chamber. The cells were cultured in ALI culture for 4 weeks, changing media every 2–3 days, until they were fully differentiated.

A co-culture system was established using 0.4-μm Transwell inserts (Corning, NY, USA) in a 6-well culture plate. For the co-culture of macrophages and cancer cells, macrophages were seeded on the upper layer and cancer cells were seeded on the bottom layer (at 60–70% confluence), and the cells were cultured for 24 h. For the co-culture of macrophages and CAFs, CAFs were seeded on the upper layer (at 30–40% confluence) and macrophages were seeded on the bottom layer, and the cells were cultured for 3 days.

HUVECs monolayer integrity was analyzed by TEER. Resistance was measured across the monolayer as described previously [20 (link)]. Briefly, 0.4 μM transwell inserts (Corning CoStar cooperation) were coated with collagen. HUVECs were plated at a density of 1 × 10⁵ cells/well. TEER was measured as described previously [21 (link),22 (link)]. Monolayers were treated with DMSO (control) for 12 h, TNF-α for 12 h, RVX208/JQ1 for 12 h, and pretreatment with RVX208/JQ1 for 12 h followed by TNF-α for 12 h. TEER was measured after 12 h treatments using the epithelial Volt/Ohm meter (EVOM2). Values were normalized against the resistance of a blank well containing only medium and coated with collagen.

The basal side of 0.4 μm transwell inserts (Corning Life Sciences, Montreal, QC, Canada) were coated with the GelTrex LDEV-free reduced growth factor basement membrane matrix (ThermoFisher Scientific, Mississauga, ON, Canada) and rested basal side up for 1 h at 37 °C and 5% CO₂. Transwell inserts were turned apical side up, the apical side coated with GelTrex, and rested for 1 h at 37 °C and 5% CO₂. Transwell inserts were turned basal side up, the basal side of the transwell inserts seeded with HPMEC at a concentration of 1.5 × 10⁵ cells/mL (4.5 × 10⁴ cells/cm²) in a 1:1 mix of HPAEpiC and HPMEC media, and rested for 3 h at 37 °C and 5% CO₂. Transwell inserts were turned apical side up and HPAEpiC were seeded on the apical side at a concentration of 3 × 10⁵ cells/mL (9 × 10⁴ cells/cm²) in a 1:1 mix of HPAEpiC and HPMEC media. After 24 h, media was removed from the upper compartment of the transwell insert, to allow primary epithelial cells to grow at the air-liquid interface. Media in the lower compartment was refreshed with a 1:1 mix of HPAEpiC and HPMEC media. Cells were permitted to grow to confluency for 14 days, with media in the lower compartment being refreshed every second day. An alveolar-capillary barrier coculture model schematic is presented in Figure S1.