Ckx71

The Lentiviral vectors production and transduction was performed as we described previously (13 (link)). rBMSCs were randomly divided into two groups for co-culture: vector group (infected with empty vector virus), and rBMSCs/ITGA5B1 group (co-infected with ITGA5 and ITGB1 virus). The transwell co-culture system was used for HPASMC and rBMSCs co-culture, briefly, HPASMC was cultured in the lower compartment of a transwell-plate (Costar 3412, Corning Incorporated, NY, USA), treated with MCT (1 μM) for 24 h after HPASMC reached 50~60% confluence, and named for MCT-HPASMC. Each group of rBMSCs (5 days post transduction) was placed onto 0.4 μm pore size polycarbonate membranes of the upper compartment in the transwell plate. HPASMC were randomly divided into four groups: Control group (only HPASMC), Model group (only MCT-HPASMC), Vector group (MCT-HPASMC in lower compartment and co-cultured with rBMSCs/Vector), ITGA5B1 group (MCT-HPASMC in lower compartment and co-cultured with rBMSCs/ITGA5B1). The morphological changes of each group of HPASMC were investigated and photographed under light microscopy (CKX71, Olympus) after co-cultured for 72 h.

Cells were seeded at a density of 5 × 10³ cells per well in 6-well ultralow attachment plates (Corning Inc., Corning, NY, USA) at 37 °C with 5% CO₂. Cells were incubated with DMEF/F12 (1 : 1) with B-27 (Thermo Fisher Scientific, Grand Island, NY, USA), 10 ng mL⁻¹ fibroblast growth factor-basic (bFGF, R&D, Minneapolis, MN, USA), and 10 ng mL⁻¹ epidermal growth factor (EGF, Gibco, Carlsbad, CA, USA). After 10 days, tumourspheres were harvested, and then equal cells were plated to form secondary generation. The compound was added at indicated concentrations after plating for 24 h. Cells were cultured for a subsequent 10 days. The culture medium with the compound was replaced three times during incubation. The cells were visualized, and the number of spheres (>50 μm) was counted using an inverted microscope (CKX71, Olympus Corporation, Tokyo, Japan)