0.4 μm pore transwell inserts

Primary human nasal airway epithelial cells (nAECs) were grown to a ciliated phenotype on 0.4-μm pore Transwell inserts (Corning) at air-liquid interface (ALI) as described previously (3 (link)). Recombinant GFP-tagged RSV A2 strain was kindly provided by Fix et al. (29 (link)) and propagated using HEp-2 cells (multiplicity of infection [MOI], 0.1) for 3 to 5 days in Opti-MEM. Virus was purified as described previously (30 (link)), collected in bronchial epithelial basal medium (BEBM; Life Technologies), and frozen at –80°C. Aliquots of RSV were thawed immediately prior to use. For ALI culture infection, the apical surface of the ALI cultures was rinsed with medium (BEBM), and 200-μl viral inoculum (MOI, 1) in BEBM was applied to the apical surface for 1 h at 37°C and then removed (see Fig. 7). Mock wells received BEBM alone. All cells were fed basolaterally with fresh ALI medium prior to infection. The infection continued for 24 h and 72 h.

FLSs were pre-seeded on the bottom chamber of a transwell plate for 24 h. PBMCs were treated with Mito-TEMPO (Sigma-Aldrich) for 30 min, washed in PBS, and then loaded onto 0.4 μm pore transwell inserts (Corning Inc., Corning, NY, USA). The transwell chambers were incubated at 37 °C for 24 h, after which the FLSs were examined to determine mRNA levels of inflammation-related genes. The culture supernatants of FLSs were used for cytokine profiling with the Proteome Profiler array (cat# ARY022B and R&D Systems) according to the manufacturer’s instructions. Quantification of cytokine optical densities was obtained with an Amersham Imager 680 (GE Healthcare, Chicago, IL, USA) and analyzed using the Quick Spots Tool (Western Vision, Salt Lake, UT, USA). The PBMC:FLS ratio in all experiments was 10:1.

PMA-stimulated THP-1 cells were added into the upper chamber of 8 μm pore transwell inserts (Corning, NY, USA). SK-HEP-1 cells with HOMER3-AS1 overexpression or silencing were plated into the lower chamber. After culture for 48 h, THP-1 cells remaining in the upper chamber were removed. The migrated THP-1 cells were fixed, stained, and detected using a microscope. SK-HEP-1 cells with HOMER3-AS1 overexpression or silencing were plated into the upper chamber of 0.4 μm pore transwell inserts (Corning). PMA-stimulated THP-1 cells were plated into the lower chamber. After co-culture for 96 h, THP-1 cells were collected to extract RNA. Genes expression in THP-1 cells were detected by qRT-PCR. THP-1 cellular proliferation was detected by CCK-8 experiments.