2.3 | HNEC culture
HNECs were cultured in DMEM/F12 medium, containing 10% foetal bovine serum (FBS) (Gibco, USA), at 37 °C in a 5% CO2incubator. The cells were passaged for less than five generations to reduce the influence of variations in primary cells on the experimental results. All cells used for in vitro stimulation were inoculated in 6-well plates for 24 h, and cultured till 60–80% confluency was achieved. The adenovirus used to overexpress MG53 (Ad-MG53), and the si-RNA (The respective si-RNA sequences are shown in Table 2) used to knock down MG53 were purchased from GenePharma (Shanghai, China). After 6 to 24 h of transfection, the cells were transferred to fresh DMEM/F12 containing 10% FBS. After 72 h of transfection, the cells were collected separately using cleaning fluid for further tests. For the in vitro drug stimulation experiment, we added 50 nM TGF-β1 inhibitor (screened by western blot; Appendix C) and 10 ng/mL of TGF-β133-35 to the cells 24 h after plating. After 72 h of stimulation, the supernatant and cells were collected for further testing.