Co-incubation assay

A trans-membrane of 0.4 µm pore size (Corning Life Science, Pittsburgh, PA, USA) was used for the analysis of soluble factors secreted by HOTAIR overexpressing hepatoma cell lines or HOTAIR-overexpressing hepatoma cell lines. The upper chamber included PBMCs (1×10⁶ cells/500 µl) from healthy donors, and the lower chamber included HOTAIR-overexpressing hepatoma cell lines (1.0×10⁵ cells/500 µl). After 72 h co-incubation at 37°C under an atmosphere of 5% CO₂, multicolor FACS analysis was performed.